Compositions and methods for treatment of cystic fibrosis

Abstract

Gene therapy compositions and methods are provided for correcting one or more mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene in a patient, thereby treating or mitigating cystic fibrosis.

Description

[0001]SAL-017PC / 126933-5017 COMPOSITIONS AND METHODS FOR TREATMENT OF CYSTIC FIBROSIS FIELD The present disclosure relates, in part, to methods, compositions, and products for treating and / or mitigating diseases and conditions related to cystic fibrosis. CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and the benefit of U.S. Provisional Patent Application No.63 / 501,500, filed on May 11, 2023, the entire content of which are hereby incorporated herein by reference in its entirety. SEQUENCE LISTING The instant application contains a sequence listing, which has been submitted in XML format via EFS-Web. The contents of the XML copy named “SAL-017PC_126933-5017_Sequence Listing,” which was created on May 8, 2024 and is 217,088 bytes in size, the contents of which are incorporated herein by reference in their entirety. BACKGROUND Cystic fibrosis (CF) is a genetic disorder that causes problems with breathing and digestion. CF affects more than about 100,000 people worldwide. People with CF typically have mucus that is thick and sticky, which blocks airways and leads to lung damage; makes infections more likely; and prevents proteins needed for digestion from reaching the intestines, which decreases the body’s ability to absorb nutrients. CF is caused by over 360 disease-causing mutations in the CFTR gene, leading to defects in the CFTR protein, which results in the abnormally thick mucus and other symptoms that characterize the disease. There remains a need for improved compositions and methods for treating cystic fibrosis in patients and / or alleviating the symptoms of cystic fibrosis in patients. SUMMARY Accordingly, the present disclosure provides, inter alia, compositions and methods for treating CF and / or mitigating pulmonary pathology in patients with CF. In embodiments, the compositions and methods of the present disclosure make use of gene transfer constructs comprising transposon expression vectors that use sequence- or locus- specific transposition (SLST) to correct conditions in patients with CFTR gene mutations. Accordingly, in aspects, a composition is provided that comprises a gene transfer construct, comprising (a) a nucleic acid encoding a cystic fibrosis transmembrane conductance regulator (CFTR) transgene or a functional fragment thereof; and (b) a vector comprising one or more inverted terminal repeats (ITRs) or end sequences. In embodiments, the CFTR transgene is or comprises human CFTR exons 2 to 27 (CFTR2-27), or a functional fragment thereof. In embodiments, the human CFTR exons 2 to 27 is suitable for insertion into an endogenous CFTR intron 1 locus, or other intronic sites in the CFTR gene. 1 DB1 / 147031523.8 SAL-017PC / 126933-5017 In embodiments, the present disclosure provides a composition wherein: (a) the ITRs or the end sequences are those of a mobile element, optionally comprising a TTAA repetitive sequence, and / or (b) the ITRs or the end sequences flank the nucleic acid encoding the CFTR transgene, or a functional fragment thereof. In embodiments, the composition of the present disclosure further comprises a nucleic acid construct encoding an enzyme capable of performing targeted genomic integration, optionally an mRNA encoding the enzyme. In embodiments, the present disclosure provides a composition, wherein the composition is encapsulated in a lipid nanoparticle (LNP). In embodiments, the polynucleotide encoding the enzyme or variant thereof and the polynucleotide encoding the transgene are in the form of the same LNP, optionally in a co-formulation. In aspects of the present disclosure, a host cell is provided that comprises the composition in accordance with embodiments of the present disclosure. BRIEF DESCRIPTION OF DRAWINGS FIG.1 depicts a non-limiting schematic representation of the present approach to gene therapy. FIG.2 depicts a schematic representation of the CFTR locus. FIG.3A, FIG.3B, and FIG.3C depicts schematic representations of RNA helper and DNA donor constructs. FIG. 3A shows an RNA helper construct using an extrinsic DNA binding domain (e.g., without limitation, dCas, chimeric dCasX molecules, TALE, Zinc finger) to target a specific TTAA site in CFTR intron 1. FIG.3B shows DNA construct using a reporter gene (e.g., without limitation, GFP or luciferase) to test for site specific CFTR integration and lipid nanoparticle (LNP) targeting. FIG.3C shows a CFTR donor DNA containing helper-specific inverted terminal repeats (ITRs) flanking the open reading frame of CFTR exons 2-27 and a 5’-splice acceptor site (e.g. without limitation, SEQ ID NO: 12, 13, and 14) and a late SV40 poly(A) signal to terminate transcription. FIG.4 depicts a schematic representation of a construct of the disclosure. Exon 1 is spliced with the inserted CFTR exon 2 through 27 of SEQ ID NO: 12. The construct has a SV40 poly A. The CFTR splice acceptor 1 includes a T804C mutation and an M470 polymorphism. FIG.5 depicts a schematic representation of a construct of the disclosure. Exon 1 is spliced with the inserted CFTR exon 2 through 27 of SEQ ID NO: 13. The construct has a SV40 poly A. The CFTR splice acceptor 2 includes a T804C mutation and an M470 polymorphism. FIG.6 depicts a schematic representation of a construct of the disclosure. Exon 1 is spliced with the inserted CFTR exon 2 through 27 of SEQ ID NO: 14. The construct has a SV40 poly A. The CFTR splice acceptor 3 includes a T804C mutation and an M470 polymorphism. FIG.7A and FIG.7B depict two regions of intron 1 of the CFTR gene. The zinc finger and TALE sequences are in TABLE 1, TABLE 2, TABLE 3, and TABLE 4. The circle in FIG.7A shows the TTAA position targeted by the zinc finger 50 (ZF50). The circle in FIG.7B show the TTAA position targeted by the Z498 and Z504 as well as TALE pairs in the “IN” orientation ( ^ ⇐). “IN” orientation is defined as the upper strand binder 5’ to 3’ upstream of the 2 DB1 / 147031523.8 SAL-017PC / 126933-5017 TTAA with the downstream lower strand binder 3’ to 5’. “OUT” orientation is defined as the upper strand binder 3’ to 5’ upsteam of the TTAA with the downstream lower strand binder 5’-3’. FIG.8 depicts the results of GFP reporter assays in HEK293 cells using helper enzymes fused to various zinc fingers (ZF), donor, and plasmid reporters containing CFTR intron 1 regions A and B. “MLT” refers to the helper enzyme derived from Myotis lucifugus and “4x” means the amino acid linker GGSGGSGGSGGS (SEQ ID NO: 133), while “8X” means the amino acid linker GGSGGSGGSGGSGGSGGSGGSGGS (SEQ ID NO: 134); “Orig” means amino acid linker AKLAGGAPAVGGGPKAADKFAATGGS (SEQ ID NO: 224). Sequences of Zinc fingers can be found in TABLE 1. FIG.9 depicts the PCR results using a plasmid reporter assay in HEK293 cells using helper enzymes fused to various zinc fingers (ZF) and TALEs, donor, and plasmid reporters containing CFTR intron 1 regions A and B. FIG. 9 shows the arrows pointed to the expected amplicons after donor insertion at the target TTAA for zinc finger- helper fusions to Z50 (Region A, 444 bp), Z498 (Region B, 625 bp) and Z504 (Region B, 625 bp). Sequences of Zinc fingers can be found in TABLE 1. FIG.10A, FIG.10B, FIG.10C, and FIG.10D depicts Amplicon-seq data using the cell pellets from the GFP reporter assay. FIG.10A shows the TTAA position (bold) where most insertions occurred at specific CFTR intron 1 sites and the location of the nearby (12bp – 18 bp) ZF binding sites for ZF 654, 504 and 498. FIG.10B, FIG.10C, and FIG.10D show the number of reads at TTAA sites within the plasmid reporters containing CFTR intron 1 regions A and B. FIG.11 depicts a reporter assay in Saccharomyces cervisiae (yeast) to detect targeting by helper proteins fused to synthetic DNA binding proteins (e.g., ZF and TALEs). FIG.12 depicts a 1.5% agarose gel and PCR amplification strategy to identify yeast with targeted integration events at the intended TTAA. FIG.13 depicts the integration efficiency in mutant (G542X) HBE of a GFP donor element targeted to CFTR intron 1 using helper-fusions (TALE and Zinc fingers). Sequences of Zinc fingers and TALEs can be found in TABLE 1 (6 finger Zinc fingers), TABLE 3 (3 finger Zinc fingers), and TABLE 4 (TALEs).3F represents 3 finger Zinc fingers and 6F represents 6 finger Zinc fingers. FIG.14 depicts a 2.0% agarose gel after transfection of the donor element in FIG.3C (CFTR exon 2-27) with different splice acceptor sites and a ZF504 helper fusion using a plasmid reporter assay in HEK293 cells. FIG.15A and FIG.15B depict 2.0% agarose gels showing 6-finger zinc fingers (6F) and 3-finger zinc fingers (3F) targeting CFTR intron in a plasmid reporter assay. FIG.15A compares 6F ZF498, 6F ZF504, 6F ZF498 + ZF504, 3F ZF498, 3F ZF504, 3F ZF498 + ZF504 targeting of CFTR intro 1 (Table 1). FIG.15B compares 6F ZF50 and 3F ZF50 targeting of CFTR intro 1. Sequences of Zinc fingers can be found in TABLE 1 (6 finger Zinc fingers), and TABLE 3 (3 finger Zinc fingers). 3 DB1 / 147031523.8 SAL-017PC / 126933-5017 FIG.16 depicts 2.0% agarose gels showing a comparison of TALE binders flanking the TTAA site targeted by ZF498 and ZF504. FIG.17A and FIG.17B depict RT-qPCR and western blot (WB), in a mutant (G542X) 16HBE cell line. FIG.18 shows a non-limiting schematic of site-specific insertion of CFTR2-27cDNA in intron 1 using a bioengineered mammalian transposase derived from Myotis lucifugus (bMLT). FIG.19A - 19C show data demonstrating dual ZnF binding sites flanking the TTAA site synergistically increasing on-target integration of desired cargo. FIG.19A shows a schematic of FiCAT Reporter Assay. FIG.19B shows a gel electrophoresis of PCR products demonstrating targeted integration. FIG.19C shows graphs demonstrating Next Generation Sequencing of integration showing 2-fold increase in targeted integrations when the target TTAA site (arrow) is flanked by two ZnF binding sites (bottom) compared to a single ZnF binding site (top). FIG.20A – 20D show data demonstrating Exc+ / Intreducedmutants increase targeted integration. FIG.20A shows a graph demonstrating mutation of non-specific target DNA binding domains of bMLT reduces integration (bars on the right) but not excision activity (bars on the left). FIG. 20B shows a graph demonstrating Next Generation Sequencing analysis showing increased targeted integration with various bMLT mutants at an engineered target site (bars on the right) within a reporter plasmid. FIG.20C shows a graph demonstrating flow analysis showing increased targeted integration at an engineered site (bars on the right) in the genome by various bMLT mutants using stable cell lines. FIG.20D shows a graph demonstrating ddPCR analysis of targeted and total integration of 1st and 2nd Generation bMLT mutants. Further mutations to bMLT (brackets) reduced off-target integration 3-4-fold (bars on the right) without affecting on-target integration (bars on the left) compared to their parent mutants. FIGs.21A - 21B show data demonstrating addition of Exc+ / Intreducedmut1 to ZnFCFTRincreases specific targeting at CFTR intron 1. FIG.21A shows a graph of Next Generation Sequencing demonstrating 40% higher on-target integration with ZnFCFTR-mut1 (bars labeled as C) than ZnFCFTR-bMLT (bars labeled as B) and 63% higher on- target integration than unfused bMLT (bars labeled as A). For orientation purposes, from left to right, the bars represent B, A, B, C, A, B, A, B, B, C, B, C, B, C, B, C, B, C, B, B, A, B, A, A, B, A, B, C, A, B, A, B, A, B, A, B, C, A, B, A, B, C, A, B, C, A, B, and B. FIG.21B shows SRT results showing a ~200-fold increase in on-target integration (bar on the left) and ~30-fold decrease in off-target integration (bars on the right). FIGs.22A – 22B show data demonstrating the addition of ZnF binding sites flanking the cargo element increases overall integration. FIG.22A shows a schematic of donor plasmids contain no flanking ZnF binding sites (top) or a ZnFCFTRbinding site flanking each ITR outside of the cargo (bottom). FIG.22B shows a flow analysis showing a ~2.5-fold increase in targeted integration by ZnFCFTR-bMLT when two flanking ZnFCFTRbinding sites are added to the cargo element. For orientation purposes, bars on the left represent promoterless GFP and bars on the right represent promoterless GFP + ZnFCFTRsites. 4 DB1 / 147031523.8 SAL-017PC / 126933-5017 FIGs.23A – 23E show data demonstrating ZnFCFTR-mut1 bMLT shows targeted genomic integration of reporter and CFTR2-27donors in HEK293T and human bronchial epithelial (HBE) cells modeling Cystic Fibrosis. FIG.23A shows a schematic of promoterless GFP and CFTR2-27-GFP plasmids with different splice acceptors. FIG.23B shows a graph demonstrating promoterless GFP and CFTR2-27-GFP plasmids with different splice acceptors were transfected alongside ZNFCFTR–mut1 bMLT into HEK293T cells under varying conditions. FIG.23C shows a schematic of targeted integration in HEK293T cells. FIG.23D shows a FACS analysis of targeted integration in HEK293T cells. Cells transfected under engineered conditions displayed higher targeted integration (up to 10%) when compared to control. FIG.23E shows a graph demonstrating targeted integration in CFTR G542X 16HBEge cells. Cells were transfected with the donor shown in FIG.23A and various Exc+ / Intreducedmutants. FACS analysis shows an up to 4-fold increase in targeted integration compared to donor alone. DETAILED DESCRIPTION The present disclosure is based, in part, on the discovery of certain gene therapy methods and compositions can be used for treating and mitigating cystic fibrosis (CF). The gene therapy methods in accordance with the present disclosure, in embodiments, find use in correcting mutations in cystic fibrosis transmembrane conductance regulator (CFTR) gene. The described methods and compositions, in embodiments, employ transposition of CFTR. The described methods and compositions, in embodiments, improve pulmonary pathology of cystic fibrosis in patients and thereby improve overall prognosis and health of the patients. The CFTR protein is a chloride channel that is expressed in many tissues. In the lung, CFTR functions to regulate the volume of airway surface liquid. Absence of functional CFTR protein results in thickened airway mucus that leads to chronic airway infection and, ultimately, severe bronchiectasis. Most CF patients die at an early age as a result of respiratory failure. Ostrowski, Lawrence E., et al. Targeting expression of a transgene to the airway surface epithelium using a ciliated cell-specific promoter. Mol. Ther.8(4), 637-645. Introducing a vector coding for wild- type CFTR into CF airway cells may be able to correct the basic defect in chloride secretion. Thus, gene therapy may be useful to treat individuals afflicted with cystic fibrosis. CF is caused by over 360 disease-causing variants in the CFTR gene. See Lee, Jin-A., et al. (2021). Gene therapy for cystic fibrosis: new tools for precision medicine. J. Transl. Med.19(1), 1-15. Construct In embodiments, the composition of the present disclosure comprises a gene transfer construct, comprising: (a) a nucleic acid encoding a cystic fibrosis transmembrane conductance regulator (CFTR) transgene or a functional fragment thereof; and (b) a vector comprising one or more inverted terminal repeats (ITRs) or end sequences. In embodiments, the gene transfer construct comprises DNA. In embodiments, the gene transfer construct is codon optimized. In embodiments, the CFTR transgene is or comprises human CFTR exons 2 to 27 (CFTR2-27), or a functional fragment thereof. In embodiments, the human CFTR exons 2 to 27 is suitable for insertion into a locus of TABLE 5 DB1 / 147031523.8 SAL-017PC / 126933-5017 B, optionally an endogenous CFTR intron 1 locus, or optionally in other intronic sites in the CFTR gene. In embodiments, the hg38 coordinates are chr7:117,480,146-117,504,250 (24,105 base pairs). In embodiments, the CFTR transgene is or comprises human CFTR exons 3 to 27 (CFTR3-27), or a functional fragment thereof. In embodiments, the CFTR transgene is or comprises human CFTR exons 4 to 27 (CFTR4-27), or a functional fragment thereof. In embodiments, the CFTR transgene is or comprises human CFTR exons 5 to 27 (CFTR5-27), or a functional fragment thereof. In embodiments, the CFTR transgene is or comprises human CFTR exons 6 to 27 (CFTR6-27), or a functional fragment thereof. In embodiments, the CFTR transgene is or comprises human CFTR exons 7 to 27 (CFTR7-27), or a functional fragment thereof. In embodiments, the CFTR gene comprises CFTR introns provided in TABLE A. TABLE A. List of CFTR introns. CFTR Intron 1-2 In embodiments, the present methods or compositions allow for intronic integration at one or more intronic integration target sites in the human CFTR gene provided in TABLE B. TABLE B. Potential intronic integration target sites in the human CFTR gene. 6 DB1 / 147031523.8 SAL-017PC / 126933-5017 CFTR Intron No. TTAA Size (bp) TTAA / bp 1-2 233 24,105 0.97% In embodiments, the composition comprises a 5’-splice acceptor site. In embodiments, the composition comprises a late SV40 poly(A) signal. CFTR is located on chromosome 7 and its genomic structure spans 189 kb including 27 exons. The CFTR open reading frame is from nucleotide position 71 to nucleotide position 4513 of SEQ ID NO: 10. The human CFTR DNA sequence (NCBI Reference: NM_000492.4) is: 1 gtagtaggtc tttggcatta ggagcttgag cccagacggc cctagcaggg accccagcgc 61 ccgagagacc atgcagaggt cgcctctgga aaaggccagc gttgtctcca aacttttttt 121 cagctggacc agaccaattt tgaggaaagg atacagacag cgcctggaat tgtcagacat 181 ataccaaatc ccttctgttg attctgctga caatctatct gaaaaattgg aaagagaatg 241 ggatagagag ctggcttcaa agaaaaatcc taaactcatt aatgcccttc ggcgatgttt 301 tttctggaga tttatgttct atggaatctt tttatattta ggggaagtca ccaaagcagt 361 acagcctctc ttactgggaa gaatcatagc ttcctatgac ccggataaca aggaggaacg 421 ctctatcgcg atttatctag gcataggctt atgccttctc tttattgtga ggacactgct 481 cctacaccca gccatttttg gccttcatca cattggaatg cagatgagaa tagctatgtt 541 tagtttgatt tataagaaga ctttaaagct gtcaagccgt gttctagata aaataagtat 7 DB1 / 147031523.8 SAL-017PC / 126933-5017 601 tggacaactt gttagtctcc tttccaacaa cctgaacaaa tttgatgaag gacttgcatt 661 ggcacatttc gtgtggatcg ctcctttgca agtggcactc ctcatggggc taatctggga 721 gttgttacag gcgtctgcct tctgtggact tggtttcctg atagtccttg ccctttttca 781 ggctgggcta gggagaatga tgatgaagta cagagatcag agagctggga agatcagtga 841 aagacttgtg attacctcag aaatgattga aaatatccaa tctgttaagg catactgctg 901 ggaagaagca atggaaaaaa tgattgaaaa cttaagacaa acagaactga aactgactcg 961 gaaggcagcc tatgtgagat acttcaatag ctcagccttc ttcttctcag ggttctttgt 1021 ggtgttttta tctgtgcttc cctatgcact aatcaaagga atcatcctcc ggaaaatatt 1081 caccaccatc tcattctgca ttgttctgcg catggcggtc actcggcaat ttccctgggc 1141 tgtacaaaca tggtatgact ctcttggagc aataaacaaa atacaggatt tcttacaaaa 1201 gcaagaatat aagacattgg aatataactt aacgactaca gaagtagtga tggagaatgt 1261 aacagccttc tgggaggagg gatttgggga attatttgag aaagcaaaac aaaacaataa 1321 caatagaaaa acttctaatg gtgatgacag cctcttcttc agtaatttct cacttcttgg 1381 tactcctgtc ctgaaagata ttaatttcaa gatagaaaga ggacagttgt tggcggttgc 1441 tggatccact ggagcaggca agacttcact tctaatggtg attatgggag aactggagcc 1501 ttcagagggt aaaattaagc acagtggaag aatttcattc tgttctcagt tttcctggat 1561 tatgcctggc accattaaag aaaatatcat ctttggtgtt tcctatgatg aatatagata 1621 cagaagcgtc atcaaagcat gccaactaga agaggacatc tccaagtttg cagagaaaga 1681 caatatagtt cttggagaag gtggaatcac actgagtgga ggtcaacgag caagaatttc 1741 tttagcaaga gcagtataca aagatgctga tttgtattta ttagactctc cttttggata 1801 cctagatgtt ttaacagaaa aagaaatatt tgaaagctgt gtctgtaaac tgatggctaa 1861 caaaactagg attttggtca cttctaaaat ggaacattta aagaaagctg acaaaatatt 1921 aattttgcat gaaggtagca gctattttta tgggacattt tcagaactcc aaaatctaca 1981 gccagacttt agctcaaaac tcatgggatg tgattctttc gaccaattta gtgcagaaag 2041 aagaaattca atcctaactg agaccttaca ccgtttctca ttagaaggag atgctcctgt 2101 ctcctggaca gaaacaaaaa aacaatcttt taaacagact ggagagtttg gggaaaaaag 2161 gaagaattct attctcaatc caatcaactc tatacgaaaa ttttccattg tgcaaaagac 2221 tcccttacaa atgaatggca tcgaagagga ttctgatgag cctttagaga gaaggctgtc 2281 cttagtacca gattctgagc agggagaggc gatactgcct cgcatcagcg tgatcagcac 2341 tggccccacg cttcaggcac gaaggaggca gtctgtcctg aacctgatga cacactcagt 2401 taaccaaggt cagaacattc accgaaagac aacagcatcc acacgaaaag tgtcactggc 2461 ccctcaggca aacttgactg aactggatat atattcaaga aggttatctc aagaaactgg 2521 cttggaaata agtgaagaaa ttaacgaaga agacttaaag gagtgctttt ttgatgatat 2581 ggagagcata ccagcagtga ctacatggaa cacatacctt cgatatatta ctgtccacaa 2641 gagcttaatt tttgtgctaa tttggtgctt agtaattttt ctggcagagg tggctgcttc 2701 tttggttgtg ctgtggctcc ttggaaacac tcctcttcaa gacaaaggga atagtactca 2761 tagtagaaat aacagctatg cagtgattat caccagcacc agttcgtatt atgtgtttta 2821 catttacgtg ggagtagccg acactttgct tgctatggga ttcttcagag gtctaccact 2881 ggtgcatact ctaatcacag tgtcgaaaat tttacaccac aaaatgttac attctgttct 2941 tcaagcacct atgtcaaccc tcaacacgtt gaaagcaggt gggattctta atagattctc 3001 caaagatata gcaattttgg atgaccttct gcctcttacc atatttgact tcatccagtt 3061 gttattaatt gtgattggag ctatagcagt tgtcgcagtt ttacaaccct acatctttgt 3121 tgcaacagtg ccagtgatag tggcttttat tatgttgaga gcatatttcc tccaaacctc 3181 acagcaactc aaacaactgg aatctgaagg caggagtcca attttcactc atcttgttac 3241 aagcttaaaa ggactatgga cacttcgtgc cttcggacgg cagccttact ttgaaactct 3301 gttccacaaa gctctgaatt tacatactgc caactggttc ttgtacctgt caacactgcg 3361 ctggttccaa atgagaatag aaatgatttt tgtcatcttc ttcattgctg ttaccttcat 3421 ttccatttta acaacaggag aaggagaagg aagagttggt attatcctga ctttagccat 3481 gaatatcatg agtacattgc agtgggctgt aaactccagc atagatgtgg atagcttgat 3541 gcgatctgtg agccgagtct ttaagttcat tgacatgcca acagaaggta aacctaccaa 3601 gtcaaccaaa ccatacaaga atggccaact ctcgaaagtt atgattattg agaattcaca 3661 cgtgaagaaa gatgacatct ggccctcagg gggccaaatg actgtcaaag atctcacagc 3721 aaaatacaca gaaggtggaa atgccatatt agagaacatt tccttctcaa taagtcctgg 3781 ccagagggtg ggcctcttgg gaagaactgg atcagggaag agtactttgt tatcagcttt 3841 tttgagacta ctgaacactg aaggagaaat ccagatcgat ggtgtgtctt gggattcaat 3901 aactttgcaa cagtggagga aagcctttgg agtgatacca cagaaagtat ttattttttc 3961 tggaacattt agaaaaaact tggatcccta tgaacagtgg agtgatcaag aaatatggaa 4021 agttgcagat gaggttgggc tcagatctgt gatagaacag tttcctggga agcttgactt 4081 tgtccttgtg gatgggggct gtgtcctaag ccatggccac aagcagttga tgtgcttggc 4141 tagatctgtt ctcagtaagg cgaagatctt gctgcttgat gaacccagtg ctcatttgga 8 DB1 / 147031523.8 SAL-017PC / 126933-5017 4201 tccagtaaca taccaaataa ttagaagaac tctaaaacaa gcatttgctg attgcacagt 4261 aattctctgt gaacacagga tagaagcaat gctggaatgc caacaatttt tggtcataga 4321 agagaacaaa gtgcggcagt acgattccat ccagaaactg ctgaacgaga ggagcctctt 4381 ccggcaagcc atcagcccct ccgacagggt gaagctcttt ccccaccgga actcaagcaa 4441 gtgcaagtct aagccccaga ttgctgctct gaaagaggag acagaagaag aggtgcaaga 4501 tacaaggctt tagagagcag cataaatgtt gacatgggac atttgctcat ggaattggag 4561 ctcgtgggac agtcacctca tggaattgga gctcgtggaa cagttacctc tgcctcagaa 4621 aacaaggatg aattaagttt ttttttaaaa aagaaacatt tggtaagggg aattgaggac 4681 actgatatgg gtcttgataa atggcttcct ggcaatagtc aaattgtgtg aaaggtactt 4741 caaatccttg aagatttacc acttgtgttt tgcaagccag attttcctga aaacccttgc 4801 catgtgctag taattggaaa ggcagctcta aatgtcaatc agcctagttg atcagcttat 4861 tgtctagtga aactcgttaa tttgtagtgt tggagaagaa ctgaaatcat acttcttagg 4921 gttatgatta agtaatgata actggaaact tcagcggttt atataagctt gtattccttt 4981 ttctctcctc tccccatgat gtttagaaac acaactatat tgtttgctaa gcattccaac 5041 tatctcattt ccaagcaagt attagaatac cacaggaacc acaagactgc acatcaaaat 5101 atgccccatt caacatctag tgagcagtca ggaaagagaa cttccagatc ctggaaatca 5161 gggttagtat tgtccaggtc taccaaaaat ctcaatattt cagataatca caatacatcc 5221 cttacctggg aaagggctgt tataatcttt cacaggggac aggatggttc ccttgatgaa 5281 gaagttgata tgccttttcc caactccaga aagtgacaag ctcacagacc tttgaactag 5341 agtttagctg gaaaagtatg ttagtgcaaa ttgtcacagg acagcccttc tttccacaga 5401 agctccaggt agagggtgtg taagtagata ggccatgggc actgtgggta gacacacatg 5461 aagtccaagc atttagatgt ataggttgat ggtggtatgt tttcaggcta gatgtatgta 5521 cttcatgctg tctacactaa gagagaatga gagacacact gaagaagcac caatcatgaa 5581 ttagttttat atgcttctgt tttataattt tgtgaagcaa aattttttct ctaggaaata 5641 tttattttaa taatgtttca aacatatata acaatgctgt attttaaaag aatgattatg 5701 aattacattt gtataaaata atttttatat ttgaaatatt gactttttat ggcactagta 5761 tttctatgaa atattatgtt aaaactggga caggggagaa cctagggtga tattaaccag 5821 gggccatgaa tcaccttttg gtctggaggg aagccttggg gctgatgcag ttgttgccca 5881 cagctgtatg attcccagcc agcacagcct cttagatgca gttctgaaga agatggtacc 5941 accagtctga ctgtttccat caagggtaca ctgccttctc aactccaaac tgactcttaa 6001 gaagactgca ttatatttat tactgtaaga aaatatcact tgtcaataaa atccatacat 6061 ttgtgtgaaa (SEQ ID NO: 10) CF disease-causing variants have been categorized into 7 classes based on CFTR protein dysfunction and / or gene expression. Class I are protein production variants that result in no functional CFTR protein with roughly 22% of CF patients harboring at least 1 mutant allele; Class II are protein processing variants that create misfolded CFTR protein and reduced expression on the cell membrane to function. Approximately 88% of CF patients have at least 1 mutant allele and the main variant p.Phe508del (F508del)-CFTR caused by a mutational deletion of the amino acid phenylalanine at the position 508 of the protein; Class III are gating variants that impair CFTR gate opening and encompasses roughly 6% of CF patients; Class IV result in defective ion channel conduction and approximately 6% of CF patients harbor this variant; Class V are insufficient protein variants and results in a reduced amount of CFTR at the surface membrane capturing 5% of CF patients; Class VI affects the stability of CFTR that causes a reduction in membrane retention and 5% of CF patients harbor at least one allele of this variant, and Class VII affects CFTR mRNA expression resulting in no mRNA and includes large deletions such as the dele2,3(21 Kb) mutation. See Lee, Jin-A., et al. (2021). Gene therapy for cystic fibrosis: new tools for precision medicine. J. Transl. Med.19(1), 1-15. In embodiments, the nucleic acid encoding CFTR transgene, or the functional fragment thereof comprises a nucleotide sequence of SEQ ID NO: 10, or a variant having at least about 90%, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto. 9 DB1 / 147031523.8 SAL-017PC / 126933-5017 In embodiments, the nucleic acid encoding CFTR transgene, or the functional fragment thereof comprises a nucleotide sequence from about nucleotide position 71 to about nucleotide position 4513 of SEQ ID NO: 10, or a variant having at least about 90%, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto. In embodiments, the nucleic acid encoding the human CFTR protein, or a functional fragment thereof comprises a nucleotide sequence of NM_000492.4 (SEQ ID NO: 10), or a variant having at least about 90%, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98% identity thereto. In embodiments, the CFTR protein is human CFTR protein, or a functional fragment thereof. In embodiments, the nucleic acid encoding the human CFTR protein, or the functional fragment thereof comprises a nucleotide sequence encoding a protein having an amino acid sequence of SEQ ID NO: 11, or a variant having at least about 90%, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98% identity thereto. MQRSPLEKASVVSKLFFSWTRPILRKGYRQRLELSDIYQIPSVDSADNLSEKLEREWDRELAS KKNPKLINALRRCFFWRFMFYGIFLYLGEVTKAVQPLLLGRIIASYDPDNKEERSIAIYLGIGLCL LFIVRTLLLHPAIFGLHHIGMQMRIAMFSLIYKKTLKLSSRVLDKISIGQLVSLLSNNLNKFDEGLA LAHFVWIAPLQVALLMGLIWELLQASAFCGLGFLIVLALFQAGLGRMMMKYRDQRAGKISERL VITSEMIENIQSVKAYCWEEAMEKMIENLRQTELKLTRKAAYVRYFNSSAFFFSGFFVVFLSVL PYALIKGIILRKIFTTISFCIVLRMAVTRQFPWAVQTWYDSLGAINKIQDFLQKQEYKTLEYNLTT TEVVMENVTAFWEEGFGELFEKAKQNNNNRKTSNGDDSLFFSNFSLLGTPVLKDINFKIERGQ LLAVAGSTGAGKTSLLMMIMGELEPSEGKIKHSGRISFCSQFSWIMPGTIKENIIFGVSYDEYRY RSVIKACQLEEDISKFAEKDNIVLGEGGITLSGGQRARISLARAVYKDADLYLLDSPFGYLDVLT EKEIFESCVCKLMANKTRILVTSKMEHLKKADKILILHEGSSYFYGTFSELQNLQPDFSSKLMG CDSFDQFSAERRNSILTETLHRFSLEGDAPVSWTETKKQSFKQTGEFGEKRKNSILNPINSIRK FSIVQKTPLQMNGIEEDSDEPLERRLSLVPDSEQGEAILPRISVISTGPTLQARRRQSVLNLMT HSVNQGQNIHRKTTASTRKVSLAPQANLTELDIYSRRLSQETGLEISEEINEEDLKECFFDDME SIPAVTTWNTYLRYITVHKSLIFVLIWCLVIFLAEVAASLVVLWLLGNTPLQDKGNSTHSRNNSY AVIITSTSSYYVFYIYVGVADTLLAMGFFRGLPLVHTLITVSKILHHKMLHSVLQAPMSTLNTLKA GGILNRFSKDIAILDDLLPLTIFDFIQLLLIVIGAIAVVAVLQPYIFVATVPVIVAFIMLRAYFLQTSQ QLKQLESEGRSPIFTHLVTSLKGLWTLRAFGRQPYFETLFHKALNLHTANWFLYLSTLRWFQM RIEMIFVIFFIAVTFISILTTGEGEGRVGIILTLAMNIMSTLQWAVNSSIDVDSLMRSVSRVFKFID MPTEGKPTKSTKPYKNGQLSKVMIIENSHVKKDDIWPSGGQMTVKDLTAKYTEGGNAILENIS FSISPGQRVGLLGRTGSGKSTLLSAFLRLLNTEGEIQIDGVSWDSITLQQWRKAFGVIPQKVFIF SGTFRKNLDPYEQWSDQEIWKVADEVGLRSVIEQFPGKLDFVLVDGGCVLSHGHKQLMCLA RSVLSKAKILLLDEPSAHLDPVTYQIIRRTLKQAFADCTVILCEHRIEAMLECQQFLVIEENKVRQ YDSIQKLLNERSLFRQAISPSDRVKLFPHRNSSKCKSKPQIAALKEETEEEVQDTRL (SEQ ID NO: 11) In embodiments, the nucleic acid encoding the human CFTR protein, or the functional fragment thereof comprises a nucleotide sequence encoding a protein having an amino acid sequence of SEQ ID NO: 306, or a variant having at least about 90%, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98% identity thereto. In embodiments, the CFTR transgene is a truncated functional fragment of the human CFTR protein. In embodiments, the CFTR transgene, or functional fragment thereof comprises exons 2-27. 10 DB1 / 147031523.8 SAL-017PC / 126933-5017 WTRPILRKGYRQRLELSDIYQIPSVDSADNLSEKLEREWDRELASKKNPKLINALRRCFFWRFMFYGIFLYLGEVT KAVQPLLLGRIIASYDPDNKEERSIAIYLGIGLCLLFIVRTLLLHPAIFGLHHIGMQMRIAMFSLIYKKTLKLSSRVLDKI SIGQLVSLLSNNLNKFDEGLALAHFVWIAPLQVALLMGLIWELLQASAFCGLGFLIVLALFQAGLGRMMMKYRDQ RAGKISERLVITSEMIENIQSVKAYCWEEAMEKMIENLRQTELKLTRKAAYVRYFNSSAFFFSGFFVVFLSVLPYAL IKGIILRKIFTTISFCIVLRMAVTRQFPWAVQTWYDSLGAINKIQDFLQKQEYKTLEYNLTTTEVVMENVTAFWEEGF GELFEKAKQNNNNRKTSNGDDSLFFSNFSLLGTPVLKDINFKIERGQLLAVAGSTGAGKTSLLMMIMGELEPSEG KIKHSGRISFCSQFSWIMPGTIKENIIFGVSYDEYRYRSVIKACQLEEDISKFAEKDNIVLGEGGITLSGGQRARISLA RAVYKDADLYLLDSPFGYLDVLTEKEIFESCVCKLMANKTRILVTSKMEHLKKADKILILHEGSSYFYGTFSELQNL QPDFSSKLMGCDSFDQFSAERRNSILTETLHRFSLEGDAPVSWTETKKQSFKQTGEFGEKRKNSILNPINSIRKF SIVQKTPLQMNGIEEDSDEPLERRLSLVPDSEQGEAILPRISVISTGPTLQARRRQSVLNLMTHSVNQGQNIHRKT TASTRKVSLAPQANLTELDIYSRRLSQETGLEISEEINEEDLKECFFDDMESIPAVTTWNTYLRYITVHKSLIFVLIW CLVIFLAEVAASLVVLWLLGNTPLQDKGNSTHSRNNSYAVIITSTSSYYVFYIYVGVADTLLAMGFFRGLPLVHTLIT VSKILHHKMLHSVLQAPMSTLNTLKAGGILNRFSKDIAILDDLLPLTIFDFIQLLLIVIGAIAVVAVLQPYIFVATVPVIVA FIMLRAYFLQTSQQLKQLESEGRSPIFTHLVTSLKGLWTLRAFGRQPYFETLFHKALNLHTANWFLYLSTLRWFQ MRIEMIFVIFFIAVTFISILTTGEGEGRVGIILTLAMNIMSTLQWAVNSSIDVDSLMRSVSRVFKFIDMPTEGKPTKST KPYKNGQLSKVMIIENSHVKKDDIWPSGGQMTVKDLTAKYTEGGNAILENISFSISPGQRVGLLGRTGSGKSTLLS AFLRLLNTEGEIQIDGVSWDSITLQQWRKAFGVIPQKVFIFSGTFRKNLDPYEQWSDQEIWKVADEVGLRSVIEQF PGKLDFVLVDGGCVLSHGHKQLMCLARSVLSKAKILLLDEPSAHLDPVTYQIIRRTLKQAFADCTVILCEHRIEAML ECQQFLVIEENKVRQYDSIQKLLNERSLFRQAISPSDRVKLFPHRNSSKCKSKPQIAALKEETEEEVQDTRL (SEQ ID NO: 306) In embodiments, the nucleotide sequence of the CFTR splice acceptor 1 is SEQ ID NO: 12. This sequence corresponds to the nucleotide sequence between the left and right ITRs in the vector shown in FIG.4. In embodiments, SEQ ID NO: 12 comprises a nucleotide polymorphism at a position corresponding to T804C. In embodiments, SEQ ID NO: 12 comprises an amino acid substitution at a position corresponding to V470M. In embodiments, the nucleic acid encoding CFTR transgene, or the functional fragment thereof comprises a nucleotide sequence of SEQ ID NO: 12, or a variant having at least about 90%, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto. SEQ ID NO 12. CFTR Splice Acceptor 1 DNA nucleotide sequence 25 bp. ctgacctcttctcttcctcccacag In embodiments, the nucleotide sequence of the CFTR splice acceptor 2 is SEQ ID NO: 13. This sequence corresponds to the nucleotide sequence between the left and right ITRs in the vector shown in FIG.5. In embodiments, SEQ ID NO: 13 comprises a nucleotide polymorphism at a position corresponding to T804C. In embodiments, SEQ ID NO: 13 comprises an amino acid substitution at a position corresponding to V470M. In embodiments, the nucleic acid encoding CFTR transgene, or the functional fragment thereof comprises a 11 DB1 / 147031523.8 SAL-017PC / 126933-5017 nucleotide sequence of SEQ ID NO: 13, or a variant having at least about 90%, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto. SEQ ID NO 13. CFTR Splice Acceptor 2 DNA nucleotide sequence 89 bp. cccccccttttttttataaaattgtatgttatatacatatctcctgtatgtgacccatgtgcttatgac tctatttctcatgtgtttag In embodiments, the nucleotide sequence of the CFTR splice acceptor 3 is SEQ ID NO: 14. This sequence corresponds to the nucleotide sequence between the left and right ITRs in the vector shown in FIG.6. In embodiments, SEQ ID NO: 14 comprises a nucleotide polymorphism at a position corresponding to T804C. In embodiments, SEQ ID NO: 14 comprises an amino acid substitution at a position corresponding to V470M. In embodiments, the nucleic acid encoding CFTR transgene, or the functional fragment thereof comprises a nucleotide sequence of SEQ ID NO: 14, or a variant having at least about 90%, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto. SEQ ID NO 14. CFTR Splice Acceptor 3 DNA nucleotide sequence 436 bp. gcaagagttccagccgggctatttacttttgtaaaactttatggtttgtggaaaacaaatgtttttgaa catttaaaaagttcagatgttaaaaagttgaaaggttaatgtaaaacaatcaatattaaagaattttga tgccaaaactattagataaaaggttaatctacatccctactagaattctcatacttaactggttggtta tgtggaagaaacatactttcacaataaagagctttaggatatgatgccattttatatcactagtaggca gaccagcagacttttttttattgtgatatgggataacctaggcatactgcactgtacactctgacatat gaagtgctctagtcaagtttaactggtgtccacagaggacatggtttaactggaattcgtcaagcctct ggttctaatttctcatttgcag In embodiments, the vector is a DNA plasmid and optionally wherein the DNA plasmid comprises one or more insulator sequences that prevent or mitigate activation or inactivation of nearby genes. In embodiments, the present disclosure provides a composition wherein: (a) the ITRs or the end sequences are those of a mobile element, optionally comprising a TTAA repetitive sequence, and / or (b) the ITRs or the end sequences flank the nucleic acid encoding the CFTR transgene, or a functional fragment thereof. In embodiments, the TTAA repetitive sequence comprises ttTTAAaa (SEQ ID NO: 302) or taTTAAta (SEQ ID NO: 303) sequences. In embodiments, the CFTR transgene further comprises a promoter, optionally wherein the CFTR transgene targets a safe harbor site. In embodiments, the CFTR transgene does not comprise a promoter, optionally wherein the CFTR transgene targets the CFTR gene. End Sequences In embodiments, the construct comprises end sequences. In embodiments, the end sequences are recognized by the present enzyme. In embodiments, the ITRs or the end sequences are selected from SEQ ID NO: 6 and SEQ ID NO: 7. 12 DB1 / 147031523.8 SAL-017PC / 126933-5017 SEQ ID NO: 6: hyperactive helper Left ITR (157 bp) The left ITR retains recognition activity when the underlined nucleotides are deleted (80 bp). 1 ttaacacttg gattgcggga aacgagttaa gtcggctcgc gtgaattgcg cgtactccgc 61 gggagccgtc ttaactcggt tcatatagat ttgcggtgga gtgcgggaaa cgtgtaaact 121 cgggccgatt gtaactgcgt attaccaaat atttgtt SEQ ID NO: 7: hyperactive helper Right ITR (212 bp) The right ITR retains recognition activity when the underlined nucleotides are deleted (80 bp). 1 aattatttat gtactgaata gataaaaaaa tgtctgtgat tgaataaatt ttcatttttt 61 acacaagaaa ccgaaaattt catttcaatc gaacccatac ttcaaaagat ataggcattt 121 taaactaact ctgattttgc gcgggaaacc taaataattg cccgcgccat cttatatttt 181 ggcgggaaat tcacccgaca ccgtagtgtt aa In embodiments, the ITRs or the end sequences are selected from SEQ ID NO: 6 and SEQ ID NO: 7, or a nucleotide sequence having at least about 90% identity thereto, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto. In embodiments, the ITRs are recognized by a helper enzyme. In embodiments, the ITRs flank the CFTR construct. The end sequences may be exact or inexact repeats and may be inverted in orientation with respect to each other. The helper enzyme acts on the end sequences to thereby “cut” the CFTR transgene (along with the end sequences) from the vector and “paste,” or integrate, the transgene into a host genome. In embodiments, the vector encodes from 5’ to 3’, a first ITR sequence, a CFTR transgene, and a second ITR sequence. In embodiments, the end sequences include at least one repeat with a nucleotide sequence having at least about 90% identity to the nucleotide sequence of SEQ ID NO: 6. In embodiments, the at least one repeat from the nucleotide sequence having at least about 90% identity, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity to the nucleotide sequence of SEQ ID NO: 6 is positioned at the 5’ end of the donor. In embodiments, the end sequences can further include at least one repeat with a nucleotide sequence having at least about 90% identity, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity to the nucleotide sequence of SEQ ID NO: 7. In embodiments, the at least one repeat from the nucleotide sequence having at least about 90%, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity to the nucleotide sequence of SEQ ID NO: 7 is positioned at the 3’ end of the donor. In embodiments, the end sequences include at least one repeat with a nucleotide sequence of SEQ ID NO: 6 or having at least about 90% identity, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto and a truncation of about 60 nucleotides, about 65 nucleotides, about 70 nucleotides, or about 75 nucleotides, or about 80 nucleotides, or about 85 nucleotides, e.g. a truncation of position about 89 to about 157, or positions corresponding thereto, of SEQ ID NO: 6. In embodiments, the left ITR comprises a nucleotide sequence of TTAACACTTGGATTGCGGGAAACGAGTTAA GTCGGCTCGCGTGAATTGCGCGTACTCCGCGGGAGCCGTCTTAACTC (SEQ ID NO: 135), or a variant thereof comprising substitutions or deletions. 13 DB1 / 147031523.8 SAL-017PC / 126933-5017 In embodiments, the right ITR comprises a nucleotide sequence of CATTTCAATCGAACCCATACTTCAAAAGAT ATAGGCATTTTAAACTAACTCTGATTTTGCGCGGGAAACCTAAATAATTGCCCGCGCCATCTTATATTTT GGCGGGAAATTCACCCGACACCGTAGTGTTAA (SEQ ID NO: 136), or a variant thereof comprising substitutions or deletions. In embodiments, the ITRs or the end sequences are selected from SEQ ID NO: 135 and SEQ ID NO: 136, or a nucleotide sequence having at least about 90% identity thereto, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto. In embodiments, the end sequences include at least one repeat with a nucleotide sequence having at least about 90% identity to the nucleotide sequence of SEQ ID NO: 135. In embodiments, the at least one repeat from the nucleotide sequence having at least about 90% identity, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity to the nucleotide sequence of SEQ ID NO: 135 is positioned at the 5’ end of the donor. In embodiments, the end sequences can further include at least one repeat with a nucleotide sequence having at least about 90% identity, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity to the nucleotide sequence of SEQ ID NO: 136. In embodiments, the at least one repeat from the nucleotide sequence having at least about 90%, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity to the nucleotide sequence of SEQ ID NO: 136 is positioned at the 3’ end of the donor. Helper Enzyme In embodiments, the composition of the present disclosure further comprises a nucleic acid construct encoding an enzyme capable of performing targeted genomic integration, optionally an mRNA encoding the enzyme. In embodiments, the enzyme is a helper enzyme. In embodiments, the method of the present disclosure utilizes the helper enzyme. In embodiments, a cell can be contacted with both construct and helper enzyme. In embodiments, the enzyme is derived from Bombyx mori, Xenopus tropicalis, Trichoplusia ni, Rhinolophus ferrumequinum, Rousettus aegyptiacus, Phyllostomus discolor, Myotis myotis, Myotis lucifugus, Pteropus vampyrus, Pipistrellus kuhlii, Pan troglodytes, Molossus molossus, Homo sapiens, Eptesicus fuscus, Scalopus aquaticus, Pipistrellus pipistrellus, Crasionycteris thonglongyai, Microcebus murinus, Hipposideros galeritus, Hipposideros armiger, Murina feae, Megaderma lyra, Echinops telfairi, Balaenoptera bonaerensis, Delphinus leucas, Monodon monoceros, Kogia breviceps, Echinops telfairi, Daubentonia madagascarensis, Mizra coquereli, or one or more of the Tn1, Tn2, Tn3, Tn5, Tn7, Tn9, Tn10, Tn552, Tn903, Tn1000 / Gamma-delta, Tn / O, tnsA, tnsB, tnsC, tniQ, IS10, ISS, IS911, Minos, Sleeping beauty, piggyBac, Tol2, Mos1, Himar1, Hermes, Tol2, Minos, Tel, P-element, MuA, Ty1, Chapaev, transib, Tc1 / mariner, or Tc3 transposon system, or biologically active fragments variants thereof, inclusive of hyperactive variants, and / or is an engineered version thereof and / or wherein the enzyme specifically recognizes the ITRs or the end sequences. In embodiments, the enzyme is derived from Myotis lucifugus or Eptesicus fuscus. 14 DB1 / 147031523.8 SAL-017PC / 126933-5017 In embodiments, the enzyme comprises an amino acid sequence having at least about 80%, or at least about 83%, or at least about 85%, or at least about 87%, or at least about 90%, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 1 and optionally has an alanine residue at position 2 of SEQ ID NO: 1 or a position corresponding thereto and / or does not contain any further residues at the C terminus. SEQ ID NO: 1: 1 MAQHSDYSDD EFCADKLSNY SCDSDLENAS TSDEDSSDDE VMVRPRTLRR RRISSSSSDS 61 ESDIEGGREE WSHVDNPPVL EDFLGHQGLN TDAVINNIED AVKLFIGDDF FEFLVEESNR 121 YYNQNRNNFK LSKKSLKWKD ITPQEMKKFL GLIVLMGQVR KDRRDDYWTT EPWTETPYFG 181 KTMTRDRFRQ IWKAWHFNNN ADIVNESDRL CKVRPVLDYF VPKFINIYKP HQQLSLDEGI 241 VPWRGRLFFR VYNAGKIVKY GILVRLLCES DTGYICNMEI YCGEGKRLLE TIQTVVSPYT 301 DSWYHIYMDN YYNSVANCEA LMKNKFRICG TIRKNRGIPK DFQTISLKKG ETKFIRKNDI 361 LLQVWQSKKP VYLISSIHSA EMEESQNIDR TSKKKIVKPN ALIDYNKHMK GVDRADQYLS 421 YYSILRRTVK WTKRLAMYMI NCALFNSYAV YKSVRQRKMG FKMFLKQTAI HWLTDDIPED 481 MDIVPDLQPV PSTSGMRAKP PTSDPPCRLS MDMRKHTLQA IVGSGKKKNI LRRCRVCSVH 541 KLRSETRYMC KFCNIPLHKG ACFEKYHTLK NY In embodiments, the enzyme comprises an amino acid sequence of at least about 90% identity to SEQ ID NO: 1. In embodiments, the enzyme comprises an amino acid sequence of at least about 93% identity to SEQ ID NO: 1. In embodiments, the enzyme comprises an amino acid sequence of at least about 95% identity to SEQ ID NO: 1. In embodiments, the enzyme comprises an amino acid sequence of at least about 98% identity to SEQ ID NO: 1. In embodiments, the enzyme comprises an amino acid sequence of at least about 99% identity to SEQ ID NO: 1. In embodiments, the nucleic acid that encodes the enzyme of SEQ ID NO: 1 has a nucleotide sequence of SEQ ID NO: 2 or a codon-optimized form thereof. SEQ ID NO: 2: nucleotide sequence encoding SEQ ID NO: 1 (1719 nt) 1 ATGGCCCAGC ACAGCGACTA CCCCGACGAC GAGTTCAGAG CCGATAAGCT GAGTAACTAC 61 AGCTGCGACA GCGACCTGGA AAACGCCAGC ACATCCGACG AGGACAGCTC TGACGACGAG 121 GTGATGGTGC GGCCCAGAAC CCTGAGACGG AGAAGAATCA GCAGCTCTAG CAGCGACTCT 181 GAATCCGACA TCGAGGGCGG CCGGGAAGAG TGGAGCCACG TGGACAACCC TCCTGTTCTG 241 GAAGATTTTC TGGGCCATCA GGGCCTGAAC ACCGACGCCG TGATCAACAA CATCGAGGAT 301 GCCGTGAAGC TGTTCATAGG AGATGATTTC TTTGAGTTCC TGGTCGAGGA ATCCAACCGC 361 TATTACAACC AGAATAGAAA CAACTTCAAG CTGAGCAAGA AAAGCCTGAA GTGGAAGGAC 421 ATCACCCCTC AGGAGATGAA AAAGTTCCTG GGACTGATCG TTCTGATGGG ACAGGTGCGG 481 AAGGACAGAA GGGATGATTA CTGGACAACC GAACCTTGGA CCGAGACCCC TTACTTTGGC 541 AAGACCATGA CCAGAGACAG ATTCAGACAG ATCTGGAAAG CCTGGCACTT CAACAACAAT 601 GCTGATATCG TGAACGAGTC TGATAGACTG TGTAAAGTGC GGCCAGTGTT GGATTACTTC 661 GTGCCTAAGT TCATCAACAT CTATAAGCCT CACCAGCAGC TGAGCCTGGA TGAAGGCATC 721 GTGCCCTGGC GGGGCAGACT GTTCTTCAGA GTGTACAATG CTGGCAAGAT CGTCAAATAC 781 GGCATCCTGG TGCGCCTTCT GTGCGAGAGC GATACAGGCT ACATCTGTAA TATGGAAATC 841 TACTGCGGCG AGGGCAAAAG ACTGCTGGAA ACCATCCAGA CCGTCGTTTC CCCTTATACC 901 GACAGCTGGT ACCACATCTA CATGGACAAC TACTACAATT CTGTGGCCAA CTGCGAGGCC 961 CTGATGAAGA ACAAGTTTAG AATCTGCGGC ACAATCAGAA AAAACAGAGG CATCCCTAAG 1021 GACTTCCAGA CCATCTCTCT GAAGAAGGGC GAAACCAAGT TCATCAGAAA GAACGACATC 1081 CTGCTCCAAG TGTGGCAGTC CAAGAAACCC GTGTACCTGA TCAGCAGCAT CCATAGCGCC 1141 GAGATGGAAG AAAGCCAGAA CATCGACAGA ACAAGCAAGA AGAAGATCGT GAAGCCCAAT 1201 GCTCTGATCG ACTACAACAA GCACATGAAA GGCGTGGACC GGGCCGACCA GTACCTGTCT 1261 TATTACTCTA TCCTGAGAAG AACAGTGAAA TGGACCAAGA GACTGGCCAT GTACATGATC 1321 AATTGCGCCC TGTTCAACAG CTACGCCGTG TACAAGTCCG TGCGACAAAG AAAAATGGGA 15 DB1 / 147031523.8 SAL-017PC / 126933-5017 1381 TTCAAGATGT TCCTGAAGCA GACAGCCATC CACTGGCTGA CAGACGACAT TCCTGAGGAC 1441 ATGGACATTG TGCCAGATCT GCAACCTGTG CCCAGCACCT CTGGTATGAG AGCTAAGCCT 1501 CCCACCAGCG ATCCTCCATG TAGACTGAGC ATGGACATGC GGAAGCACAC CCTGCAGGCC 1561 ATCGTCGGCA GCGGCAAGAA GAAGAACATC CTTAGACGGT GCAGGGTGTG CAGCGTGCAC 1621 AAGCTGCGGA GCGAGACTCG GTACATGTGC AAGTTTTGCA ACATTCCCCT GCACAAGGGA 1681 GCCTGCTTCG AGAAGTACCA CACCCTGAAG AATTACTAG In embodiments, the enzyme has one or more mutations. In embodiments, the enzyme has one or more mutations which confer hyperactivity. In embodiments, the enzyme has one or more amino acid substitutions selected from S8X1and / or C13X2or substitutions at positions corresponding thereto. In embodiments, the enzyme has S8X1and C13X2substitutions or substitutions at positions corresponding thereto. In embodiments, X1is selected from G, A, V, L, I and P and X2is selected from K, R, and H. In embodiments, X1is P and X2is R. In embodiments, the helper enzyme, having gene cleavage (Exc) and / or gene integration (Int) activity, has at least about 90% identity, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity to the nucleotide sequence of SEQ ID NO: 3 or the amino acid sequence SEQ ID NO: 4. SEQ ID NO: 3: nucleotide sequence of hyperactive helper mRNA helper construct (1956 bp) (Order of underlined sequences: T7 promoter, hyperactive helper, polyA tail; the 5’-globin and 3’-globin UTRs are in capital letters). 1 taatacgact cactataagg aagCTTCTTG TTCTTTTTGC AGAAGCTCAG AATAAACGCT 61 CAACTTTGGc cgccaccatg gcccagcaca gcgactaccc cgacgacgag ttcagagccg 121 ataagctgag taactacagc tgcgacagcg acctggaaaa cgccagcaca tccgacgagg 181 acagctctga cgacgaggtg atggtgcggc ccagaaccct gagacggaga agaatcagca 241 gctctagcag cgactctgaa tccgacatcg agggcggccg ggaagagtgg agccacgtgg 301 acaaccctcc tgttctggaa gattttctgg gccatcaggg cctgaacacc gacgccgtga 361 tcaacaacat cgaggatgcc gtgaagctgt tcataggaga tgatttcttt gagttcctgg 421 tcgaggaatc caaccgctat tacaaccaga atagaaacaa cttcaagctg agcaagaaaa 481 gcctgaagtg gaaggacatc acccctcagg agatgaaaaa gttcctggga ctgatcgttc 541 tgatgggaca ggtgcggaag gacagaaggg atgattactg gacaaccgaa ccttggaccg 601 agacccctta ctttggcaag accatgacca gagacagatt cagacagatc tggaaagcct 661 ggcacttcaa caacaatgct gatatcgtga acgagtctga tagactgtgt aaagtgcggc 721 cagtgttgga ttacttcgtg cctaagttca tcaacatcta taagcctcac cagcagctga 781 gcctggatga aggcatcgtg ccctggcggg gcagactgtt cttcagagtg tacaatgctg 841 gcaagatcgt caaatacggc atcctggtgc gccttctgtg cgagagcgat acaggctaca 901 tctgtaatat ggaaatctac tgcggcgagg gcaaaagact gctggaaacc atccagaccg 961 tcgtttcccc ttataccgac agctggtacc acatctacat ggacaactac tacaattctg 1021 tggccaactg cgaggccctg atgaagaaca agtttagaat ctgcggcaca atcagaaaaa 1081 acagaggcat ccctaaggac ttccagacca tctctctgaa gaagggcgaa accaagttca 1141 tcagaaagaa cgacatcctg ctccaagtgt ggcagtccaa gaaacccgtg tacctgatca 1201 gcagcatcca tagcgccgag atggaagaaa gccagaacat cgacagaaca agcaagaaga 1261 agatcgtgaa gcccaatgct ctgatcgact acaacaagca catgaaaggc gtggaccggg 1321 ccgaccagta cctgtcttat tactctatcc tgagaagaac agtgaaatgg accaagagac 1381 tggccatgta catgatcaat tgcgccctgt tcaacagcta cgccgtgtac aagtccgtgc 1441 gacaaagaaa aatgggattc aagatgttcc tgaagcagac agccatccac tggctgacag 1501 acgacattcc tgaggacatg gacattgtgc cagatctgca acctgtgccc agcacctctg 1561 gtatgagagc taagcctccc accagcgatc ctccatgtag actgagcatg gacatgcgga 1621 agcacaccct gcaggccatc gtcggcagcg gcaagaagaa gaacatcctt agacggtgca 1681 gggtgtgcag cgtgcacaag ctgcggagcg agactcggta catgtgcaag ttttgcaaca 1741 ttcccctgca caagggagcc tgcttcgaga agtaccacac cctgaagaat tactagAACC 1801 AGCCTCAAGA ACACCCGAAT GGAGTCTCTA AGCTACATAA TACCAACTTA CACTTTACAA 16 DB1 / 147031523.8 SAL-017PC / 126933-5017 1861 AATGTTGTCC CCCAAAATGT AGCCATTCGT ATCTGCTCCT AATAAAAAGA AAGTTTCTTC 1921 ACaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaa In embodiments, the enzyme comprises an amino acid sequence of SEQ ID NO: 4. In embodiments, the enzyme comprises an amino acid sequence having at least about 90% identity, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity sequence identity to SEQ ID NO: 4. SEQ ID NO: 4: amino acid sequence of hyperactive helper (572 amino acids) 1 MAQHSDYPDD EFRADKLSNY SCDSDLENAS TSDEDSSDDE VMVRPRTLRR RRISSSSSDS 61 ESDIEGGREE WSHVDNPPVL EDFLGHQGLN TDAVINNIED AVKLFIGDDF FEFLVEESNR 121 YYNQNRNNFK LSKKSLKWKD ITPQEMKKFL GLIVLMGQVR KDRRDDYWTT EPWTETPYFG 181 KTMTRDRFRQ IWKAWHFNNN ADIVNESDRL CKVRPVLDYF VPKFINIYKP HQQLSLDEGI 241 VPWRGRLFFR VYNAGKIVKY GILVRLLCES DTGYICNMEI YCGEGKRLLE TIQTVVSPYT 301 DSWYHIYMDN YYNSVANCEA LMKNKFRICG TIRKNRGIPK DFQTISLKKG ETKFIRKNDI 361 LLQVWQSKKP VYLISSIHSA EMEESQNIDR TSKKKIVKPN ALIDYNKHMK GVDRADQYLS 421 YYSILRRTVK WTKRLAMYMI NCALFNSYAV YKSVRQRKMG FKMFLKQTAI HWLTDDIPED 481 MDIVPDLQPV PSTSGMRAKP PTSDPPCRLS MDMRKHTLQA IVGSGKKKNI LRRCRVCSVH 541 KLRSETRYMC KFCNIPLHKG ACFEKYHTLK NY In embodiments, the nucleic acid that encodes the enzyme of SEQ ID NO: 4 has a nucleotide sequence of SEQ ID NO: 5 or a codon-optimized form thereof. In embodiments, the nucleic acid comprises a nucleotide sequence having at least about 90% identity, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity sequence identity to SEQ ID NO: 5 or a codon-optimized form thereof. SEQ ID NO: 5: nucleotide sequence encoding the hyperactive helper (1719 nt) 1 ATGGCCCAGC ACAGCGACTA CCCCGACGAC GAGTTCAGAG CCGATAAGCT GAGTAACTAC 61 AGCTGCGACA GCGACCTGGA AAACGCCAGC ACATCCGACG AGGACAGCTC TGACGACGAG 121 GTGATGGTGC GGCCCAGAAC CCTGAGACGG AGAAGAATCA GCAGCTCTAG CAGCGACTCT 181 GAATCCGACA TCGAGGGCGG CCGGGAAGAG TGGAGCCACG TGGACAACCC TCCTGTTCTG 241 GAAGATTTTC TGGGCCATCA GGGCCTGAAC ACCGACGCCG TGATCAACAA CATCGAGGAT 301 GCCGTGAAGC TGTTCATAGG AGATGATTTC TTTGAGTTCC TGGTCGAGGA ATCCAACCGC 361 TATTACAACC AGAATAGAAA CAACTTCAAG CTGAGCAAGA AAAGCCTGAA GTGGAAGGAC 421 ATCACCCCTC AGGAGATGAA AAAGTTCCTG GGACTGATCG TTCTGATGGG ACAGGTGCGG 481 AAGGACAGAA GGGATGATTA CTGGACAACC GAACCTTGGA CCGAGACCCC TTACTTTGGC 541 AAGACCATGA CCAGAGACAG ATTCAGACAG ATCTGGAAAG CCTGGCACTT CAACAACAAT 601 GCTGATATCG TGAACGAGTC TGATAGACTG TGTAAAGTGC GGCCAGTGTT GGATTACTTC 661 GTGCCTAAGT TCATCAACAT CTATAAGCCT CACCAGCAGC TGAGCCTGGA TGAAGGCATC 721 GTGCCCTGGC GGGGCAGACT GTTCTTCAGA GTGTACAATG CTGGCAAGAT CGTCAAATAC 781 GGCATCCTGG TGCGCCTTCT GTGCGAGAGC GATACAGGCT ACATCTGTAA TATGGAAATC 841 TACTGCGGCG AGGGCAAAAG ACTGCTGGAA ACCATCCAGA CCGTCGTTTC CCCTTATACC 901 GACAGCTGGT ACCACATCTA CATGGACAAC TACTACAATT CTGTGGCCAA CTGCGAGGCC 961 CTGATGAAGA ACAAGTTTAG AATCTGCGGC ACAATCAGAA AAAACAGAGG CATCCCTAAG 1021 GACTTCCAGA CCATCTCTCT GAAGAAGGGC GAAACCAAGT TCATCAGAAA GAACGACATC 1081 CTGCTCCAAG TGTGGCAGTC CAAGAAACCC GTGTACCTGA TCAGCAGCAT CCATAGCGCC 1141 GAGATGGAAG AAAGCCAGAA CATCGACAGA ACAAGCAAGA AGAAGATCGT GAAGCCCAAT 1201 GCTCTGATCG ACTACAACAA GCACATGAAA GGCGTGGACC GGGCCGACCA GTACCTGTCT 1261 TATTACTCTA TCCTGAGAAG AACAGTGAAA TGGACCAAGA GACTGGCCAT GTACATGATC 1321 AATTGCGCCC TGTTCAACAG CTACGCCGTG TACAAGTCCG TGCGACAAAG AAAAATGGGA 1381 TTCAAGATGT TCCTGAAGCA GACAGCCATC CACTGGCTGA CAGACGACAT TCCTGAGGAC 1441 ATGGACATTG TGCCAGATCT GCAACCTGTG CCCAGCACCT CTGGTATGAG AGCTAAGCCT 1501 CCCACCAGCG ATCCTCCATG TAGACTGAGC ATGGACATGC GGAAGCACAC CCTGCAGGCC 1561 ATCGTCGGCA GCGGCAAGAA GAAGAACATC CTTAGACGGT GCAGGGTGTG CAGCGTGCAC 1621 AAGCTGCGGA GCGAGACTCG GTACATGTGC AAGTTTTGCA ACATTCCCCT GCACAAGGGA 1681 GCCTGCTTCG AGAAGTACCA CACCCTGAAG AATTACTAG 17 DB1 / 147031523.8 SAL-017PC / 126933-5017 In embodiments, the enzyme comprises an amino acid sequence of at least about 90% identity to SEQ ID NO: 15. In embodiments, the enzyme comprises an amino acid sequence of at least about 93% identity to SEQ ID NO: 15. In embodiments, the enzyme comprises an amino acid sequence of at least about 95% identity to SEQ ID NO: 15. In embodiments, the enzyme comprises an amino acid sequence of at least about 98% identity to SEQ ID NO: 15. In embodiments, the enzyme comprises an amino acid sequence of at least about 99% identity to SEQ ID NO: 15. In embodiments, the nucleic acid that encodes the enzyme of SEQ ID NO: 15 has a nucleotide sequence of SEQ ID NO: 16 or a codon-optimized form thereof. SEQ ID NO 15. Amino acid sequence (582 amino acids) of Saliogase “wild type” helper with NLS that when combined with shorted left ends (LE65, LE88, or LE90). Results in increase enzyme activity. 1 MAPKKKRKVG GAQHSDYSDD EFCADKLSNY SCDSDLENAS TSDEDSSDDE VMVRPRTLRR 61 RRISSSSSDS ESDIEGGREE WSHVDNPPVL EDFLGHQGLN TDAVINNIED AVKLFIGDDF 121 FEFLVEESNR YYNQNRNNFK LSKKSLKWKD ITPQEMKKFL GLIVLMGQVR KDRRDDYWTT 181 EPWTETPYFG KTMTRDRFRQ IWKAWHFNNN ADIVNESDRL CKVRPVLDYF VPKFINIYKP 241 HQQLSLDEGI VPWRGRLFFR VYNAGKIVKY GILVRLLCES DTGYICNMEI YCGEGKRLLE 301 TIQTVVSPYT DSWYHIYMDN YYNSVANCEA LMKNKFRICG TIRKNRGIPK DFQTISLKKG 361 ETKFIRKNDI LLQVWQSKKP VYLISSIHSA EMEESQNIDR TSKKKIVKPN ALIDYNKHMK 421 GVDRADQYLS YYSILRRTVK WTKRLAMYMI NCALFNSYAV YKSVRQRKMG FKMFLKQTAI 481 HWLTDDIPED MDIVPDLQPV PSTSGMRAKP PTSDPPCRLS MDMRKHTLQA IVGSGKKKNI 541 LRRCRVCSVH KLRSETRYMC KFCNIPLHKG ACFEKYHTLK NY SEQ ID NO 16. Nucleotide sequence (1746 bp) of Saliogase “wild type” helper with NLS that when combined with shorted left ends (LE65, LE88, or LE90). Results in increase enzyme activity. 1 atggcgccta agaagaagag aaaggtcggc ggcgcccagc acagcgacta ctccgacgac 61 gagttctgcg ccgataagct gagtaactac agctgcgaca gcgacctgga aaacgccagc 121 acatccgacg aggacagctc tgacgacgag gtgatggtgc ggcccagaac cctgagacgg 181 agaagaatca gcagctctag cagcgactct gaatccgaca tcgagggcgg ccgggaagag 241 tggagccacg tggacaaccc tcctgttctg gaagattttc tgggccatca gggcctgaac 301 accgacgccg tgatcaacaa catcgaggat gccgtgaagc tgttcatagg agatgatttc 361 tttgagttcc tggtcgagga atccaaccgc tattacaacc agaatagaaa caacttcaag 421 ctgagcaaga aaagcctgaa gtggaaggac atcacccctc aggagatgaa aaagttcctg 481 ggactgatcg ttctgatggg acaggtgcgg aaggacagaa gggatgatta ctggacaacc 541 gaaccttgga ccgagacccc ttactttggc aagaccatga ccagagacag attcagacag 601 atctggaaag cctggcactt caacaacaat gctgatatcg tgaacgagtc tgatagactg 661 tgtaaagtgc ggccagtgtt ggattacttc gtgcctaagt tcatcaacat ctataagcct 721 caccagcagc tgagcctgga tgaaggcatc gtgccctggc ggggcagact gttcttcaga 781 gtgtacaatg ctggcaagat cgtcaaatac ggcatcctgg tgcgccttct gtgcgagagc 841 gatacaggct acatctgtaa tatggaaatc tactgcggcg agggcaaaag actgctggaa 901 accatccaga ccgtcgtttc cccttatacc gacagctggt accacatcta catggacaac 961 tactacaatt ctgtggccaa ctgcgaggcc ctgatgaaga acaagtttag aatctgcggc 1021 acaatcagaa aaaacagagg catccctaag gacttccaga ccatctctct gaagaagggc 1081 gaaaccaagt tcatcagaaa gaacgacatc ctgctccaag tgtggcagtc caagaaaccc 1141 gtgtacctga tcagcagcat ccatagcgcc gagatggaag aaagccagaa catcgacaga 1201 acaagcaaga agaagatcgt gaagcccaat gctctgatcg actacaacaa gcacatgaaa 1261 ggcgtggacc gggccgacca gtacctgtct tattactcta tcctgagaag aacagtgaaa 1321 tggaccaaga gactggccat gtacatgatc aattgcgccc tgttcaacag ctacgccgtg 1381 tacaagtccg tgcgacaaag aaaaatggga ttcaagatgt tcctgaagca gacagccatc 1441 cactggctga cagacgacat tcctgaggac atggacattg tgccagatct gcaacctgtg 1501 cccagcacct ctggtatgag agctaagcct cccaccagcg atcctccatg tagactgagc 1561 atggacatgc ggaagcacac cctgcaggcc atcgtcggca gcggcaagaa gaagaacatc 1621 cttagacggt gcagggtgtg cagcgtgcac aagctgcgga gcgagactcg gtacatgtgc 18 DB1 / 147031523.8 SAL-017PC / 126933-5017 1681 aagttttgca acattcccct gcacaaggga gcctgcttcg agaagtacca caccctgaag 1741 aattac In embodiments, the helper enzyme comprises at least one substitution at position S8 or a position corresponding thereto relative to SEQ ID NO: 1. In embodiments, the substitution at position S8 or a position corresponding thereto relative to SEQ ID NO: 1 is a polar and neutral of charge hydrophilic residue selected from asparagine (N), glutamine (Q), serine (S), threonine (T), proline (P), and cysteine (C). In embodiments, the substitution at position S8 is proline (P). In embodiments, the helper enzyme comprises at least one substitution at position C13 or a position corresponding thereto relative to SEQ ID NO: 1. In embodiments, the substitution at position C13 or a position corresponding thereto relative to SEQ ID NO: 1 is a polar and positively charged hydrophilic residue optionally selected from arginine (R) and lysine (K). In embodiments, the substitution at position C13 or a position corresponding thereto relative to SEQ ID NO: 1 is an arginine (R). In embodiments, the helper enzyme comprises at least one substitution at position N125. or a position corresponding thereto relative to SEQ ID NO: 1. In embodiments, the substitution at position N125, or a position corresponding thereto relative to SEQ ID NO: 1, is a polar and positively charged hydrophilic residue optionally selected from arginine (R) and lysine (K). In embodiments, the substitution at position N125, or a position corresponding thereto relative to SEQ ID NO: 1, is lysine (K). In embodiments, the helper enzyme comprises at least one substitution at position D416, or a position corresponding thereto relative to SEQ ID NO: 1. In embodiments, the substitution at position D416 or a position corresponding thereto relative to SEQ ID NO: 1 is a polar and neutral of charge hydrophilic residue selected from asparagine (N), glutamine (Q), serine (S), threonine (T), proline (P), and cysteine (C). In embodiments, the substitution at position D416 or a position corresponding thereto relative to SEQ ID NO: 1 is asparagine (N). In embodiments, the helper enzyme comprises at least one substitution at position Y281, or a position corresponding thereto relative to SEQ ID NO: 1. In embodiments, the substitution at position Y281 or a position corresponding thereto relative to SEQ ID NO: 1 is non-polar aliphatic amino acid is selected from alanine (A), glycine (G), valine (V), leucine (L), isoleucine (I), methionine (M), and proline (P). In embodiments, the substitution at position Y281 or a position corresponding thereto relative to SEQ ID NO: 1 is alanine (A). In embodiments, the helper enzyme comprises at least one substitution at position G285, or a position corresponding thereto relative to SEQ ID NO: 1. In embodiments, the substitution at position G285 or a position corresponding thereto relative to SEQ ID NO: 1 is non-polar aliphatic amino acid is selected from alanine (A), glycine (G), valine (V), leucine (L), isoleucine (I), methionine (M), and proline (P). In embodiments, the substitution at position G285 or a position corresponding thereto relative to SEQ ID NO: 1 is alanine (A). In embodiments, the helper enzyme comprises at least one substitution at position I332, or a position corresponding thereto relative to SEQ ID NO: 1. In embodiments, the substitution at position I332 or a position 19 DB1 / 147031523.8 SAL-017PC / 126933-5017 corresponding thereto relative to SEQ ID NO: 1 is non-polar aliphatic amino acid is selected from alanine (A), glycine (G), valine (V), leucine (L), isoleucine (I), methionine (M), and proline (P). In embodiments, the substitution at position I332 or a position corresponding thereto relative to SEQ ID NO: 1 is methionine (M). In embodiments, the helper enzyme comprises at least one substitution at position R333, or a position corresponding thereto relative to SEQ ID NO: 1. In embodiments, the substitution at position R333, or a position corresponding thereto relative to SEQ ID NO: 1 is a polar and neutral of charge hydrophilic residue selected from asparagine (N), glutamine (Q), serine (S), threonine (T), proline (P), and cysteine (C), or a polar and positively charged hydrophilic residue selected from arginine (R) and lysine (K), or a non-polar aliphatic amino acid selected from alanine (A), glycine (G), valine (V), leucine (L), isoleucine (I), methionine (M), and proline (P). In embodiments, the substitution at position R333, or a position corresponding thereto relative to SEQ ID NO: 1 is alanine (A). In embodiments, the substitution at position R333, or a position corresponding thereto relative to SEQ ID NO: 1 is lysine (K). In embodiments, the substitution at position R333, or a position corresponding thereto relative to SEQ ID NO: 1 is serine (S). In embodiments, the helper enzyme comprises at least one substitution at position K334, or a position corresponding thereto relative to SEQ ID NO: 1. In embodiments, the substitution at position K334, or a position corresponding thereto relative to SEQ ID NO: 1 is a polar and neutral of charge hydrophilic residue selected from asparagine (N), glutamine (Q), serine (S), threonine (T), proline (P), and cysteine (C), or a polar and positively charged hydrophilic residue selected from arginine (R) and lysine (K), or a non-polar aliphatic amino acid selected from alanine (A), glycine (G), valine (V), leucine (L), isoleucine (I), methionine (M), and proline (P). In embodiments, the substitution at position K334, or a position corresponding thereto relative to SEQ ID NO: 1 is alanine (A). In embodiments, the substitution at position K334, or a position corresponding thereto relative to SEQ ID NO: 1 is arginine (R). In embodiments, the substitution at position K334, or a position corresponding thereto relative to SEQ ID NO: 1 is serine (S). In embodiments, the helper enzyme comprises at least one substitution at position N335, or a position corresponding thereto relative to SEQ ID NO: 1. In embodiments, the substitution at position N335 or a position corresponding thereto relative to SEQ ID NO: 1 is non-polar aliphatic amino acid is selected from alanine (A), glycine (G), valine (V), leucine (L), isoleucine (I), methionine (M), and proline (P). In embodiments, the substitution at position N335 or a position corresponding thereto relative to SEQ ID NO: 1 is alanine (A). In embodiments, the helper enzyme comprises at least one substitution at position R336. In embodiments, the substitution at position R336, or a position corresponding thereto relative to SEQ ID NO: 1 is a polar and neutral of charge hydrophilic residue selected from asparagine (N), glutamine (Q), serine (S), threonine (T), proline (P), and cysteine (C), or a polar and positively charged hydrophilic residue selected from arginine (R) and lysine (K), or a non-polar aliphatic amino acid selected from alanine (A), glycine (G), valine (V), leucine (L), isoleucine (I), methionine (M), and proline (P). In embodiments, the substitution at position R336, or a position corresponding thereto relative to SEQ ID NO: 1 is alanine (A). In embodiments, the substitution at position R336, or a position 20 DB1 / 147031523.8 SAL-017PC / 126933-5017 corresponding thereto relative to SEQ ID NO: 1 is lysine (K). In embodiments, the substitution at position R336, or a position corresponding thereto relative to SEQ ID NO: 1 is serine (S). In embodiments, the helper enzyme comprises at least one substitution at position G337, or a position corresponding thereto relative to SEQ ID NO: 1. In embodiments, the substitution at position G337 or a position corresponding thereto relative to SEQ ID NO: 1 is non-polar aliphatic amino acid is selected from alanine (A), glycine (G), valine (V), leucine (L), isoleucine (I), methionine (M), and proline (P). In embodiments, the substitution at position G337 or a position corresponding thereto relative to SEQ ID NO: 1 is alanine (A). In embodiments, the helper enzyme comprises at least one substitution at position I338, or a position corresponding thereto relative to SEQ ID NO: 1. In embodiments, the substitution at position I338 or a position corresponding thereto relative to SEQ ID NO: 1 is non-polar aliphatic amino acid is selected from alanine (A), glycine (G), valine (V), leucine (L), isoleucine (I), methionine (M), and proline (P). In embodiments, the substitution at position I338 or a position corresponding thereto relative to SEQ ID NO: 1 is methionine (M). In embodiments, the enzyme has one or more amino acid substitutions selected from S8X1, C13X2, and / or D416X3or substitutions corresponding thereto relative to SEQ ID NO: 1. In embodiments, X1is selected from G, A, V, L, I and P. In embodiments, X2is selected from K, R, and H. In embodiments, X3is selected from N, Q, S, T, P, and C. In embodiments, X1is P, X2is R and X3is N. In embodiments, the enzyme has at least one, or two, or three, or four, or five, or six, or seven, or eight substitutions at a position corresponding to Y281A, G285A, I332M, R333A, R333K, R333S, K334A, K334R, K334S, N335A, R336A, R336K, R336S, G337A, and I338M or substitutions corresponding thereto relative to SEQ ID NO: 1. In embodiments, the enzyme has enzyme has at least one, or two, or three, or four, or five, or six, or seven, or eight substitutions at a position corresponding to S8P, C13R, D416N Y281A, G285A, I332M, R333A, R333K, R333S, K334A, K334R, K334S, N335A, R336A, R336K, R336S, G337A, and I338M or substitutions corresponding thereto relative to SEQ ID NO: 1. In embodiments, the helper enzyme comprises a substitution selected from TABLE C and / or TABLE D or a substitution at positions corresponding thereto relative to SEQ ID NO: 1. Table C: Exemplary Mutations Mut# Mutations 21 DB1 / 147031523.8 SAL-017PC / 126933-5017 5 S8P_C13R_D416N_R333A_N335A 6 S8PC13RD416NK334AR336A M Table D: Exemplary mutants linked to E2C zinc fingers Mut# Mutations 22 DB1 / 147031523.8 SAL-017PC / 126933-5017 1014 E2C-S8P_C13R_D416N_R333A_N335A_K334A _ _ _ N_Y281A_K334A_N335A, S8P_C13R_D416N_R333A, S8P_C13R_D416N_R333A_K334A, S8P_C13R_D416N_R333A_N335A, S8P_C13R_D416N_K334A_R336A, S8P_C13R_D416N_Y281A_K334A_N335A_I332M, S8P_C13R_D416N_Y281A_K334A_N335A_I338M, S8P_C13R_D416N_Y281A_K334A_N335A_I332M_I338M, S8P_C13R_D416N_R333A_N335A_K334R, S8P_C13R_D416N_R333A_N335A_K334S, S8P_C13R_D416N_R333A_N335A_K334A, S8P_C13R_D416N_R333A_N335A_I338M, S8P_C13R_D416N_R333A_N335A_I332M_I338M, or substitutions corresponding thereto relative to SEQ ID NO: 1. In embodiments, the helper enzyme comprises E2C-S8P_C13R, E2C- S8P_C13R_D416N_Y281A_K334A_N335A, E2C-S8P_C13R_D416N_R333A, E2C- S8P_C13R_D416N_R333A_K334A, E2C-S8P_C13R_D416N_R333A_N335A, E2C- S8P_C13R_D416N_K334A_R336A, E2C-S8P_C13R_D416N_Y281A_K334A_N335A, E2C- S8P_C13R_D416N_Y281A_K334A_N335A_I332M, E2C-S8P_C13R_D416N_Y281A_K334A_N335A_I338M, E2C-S8P_C13R_D416N_Y281A_K334A_N335A_I332M_I338M, E2C-S8P_C13R_D416N_R333A_N335A, E2C- S8P_C13R_D416N_R333A_N335A_K334R, E2C-S8P_C13R_D416N_R333A_N335A_K334S, E2C- S8P_C13R_D416N_R333A_N335A_K334A, or substitutions corresponding thereto relative to SEQ ID NO: 1. In embodiments, the helper enzyme comprises an N- or C- terminal deletion, e.g. or about 30, or about 35, or about 40, or about 45, or about 50, or about 55, or about 65 amino acids, optionally at positions 2-35, or 2-45, or 2-68, or positions corresponding thereto, wherein the positions are relative to SEQ ID NO: 1. In embodiments, the helper enzyme comprises an N- or C- terminal deletion, e.g. or about 30, or about 35, or about 40, or about 45, or about 50, or about 55, or about 65 amino acids, optionally at positions 2-35, or 2-45, or 2-68, or positions corresponding thereto, wherein the positions are relative to SEQ ID NO: 1 and has a ZnF or TALE fused to the N-terminus. In embodiments, the enzyme has increased activity relative to an enzyme comprising an amino acid sequence of SEQ ID NO: 1 or functional equivalent thereof. In embodiments, the enzyme has excision activity. In embodiments, the enzyme has decreased integration activity. In embodiments, the enzyme has decreased integration activity relative to an enzyme comprising an amino acid sequence of SEQ ID NO: 1 or functional equivalent thereof. In embodiments, the enzyme has increased excision activity relative to an enzyme comprising an amino acid sequence of SEQ ID NO: 1 or functional equivalent thereof. 23 DB1 / 147031523.8 SAL-017PC / 126933-5017 In embodiments, the helper enzyme further comprises a zinc finger inserted after a residue selected from S60-E70 relative to SEQ ID NO: 1. In embodiments, the helper enzyme further comprises a zinc finger inserted after the G66 residue relative to SEQ ID NO: 1, optionally by way of a linker. In embodiments, the helper enzyme further comprises a zinc finger inserted after the S60 residue relative to SEQ ID NO: 1. In embodiments, the helper enzyme further comprises a zinc finger inserted after the E70 residue relative to SEQ ID NO: 1. In embodiments, the helper enzyme further comprises a zinc finger inserted after the L80 residue relative to SEQ ID NO: 1. In embodiments, the helper enzyme further comprises a zinc finger inserted after the N90 residue relative to SEQ ID NO: 1. In embodiments, the helper enzyme further comprises a zinc finger inserted after the D100 residue relative to SEQ ID NO: 1. In embodiments, there is provided a composition comprising a gene transfer construct, comprising: a nucleic acid encoding a cystic fibrosis transmembrane conductance regulator (CFTR) transgene or a functional fragment thereof, wherein the nucleic acid construct further comprises a nucleic acid construct encoding an enzyme capable of performing targeted genomic integration, optionally wherein the enzyme comprises an amino acid sequence having at least about 80%, or at least about 90%, or at least about 93%, or at least about 95%, or at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 1, and a vector comprising one or more inverted terminal repeats (ITRs) or end sequences. In embodiments, the enzyme comprises S8P_C13R_D416N_Y281A_K334A_N335A_I332M, or substitutions corresponding thereto relative to SEQ ID NO: 1. In embodiments, the enzyme comprises a targeting element. In embodiments, the targeting element comprises a Zinc finger, optionally a 6 finger Zinc finger or a 3 finger Zinc finger. In embodiments, the Zinc finger is a E2C Zinc finger. In embodiments, the E2C Zinc finger comprises at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% identity to an amino acid sequence of SEQ ID NO: 305 or a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 304 or a codon-optimized form thereof. Targeting Element In embodiments, the enzyme comprises a targeting element. In embodiments, wherein the targeting element binds target sites in CFTR intron 1. In embodiments, the target sites in CFTR intron 1 comprise a nucleotide sequence having at least about 80%, or at least about 90%, or at least about 93%, or at least about 95%, or at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 313. In embodiments, the targeting element binds a target site in a genomic safe harbor site (GSHS). In embodiments, the enzyme is capable of inserting a donor nucleic acid comprising a transgene in the CFTR gene in an intronic region (e.g., intron 1). In embodiments, the enzyme associated with the targeting element, is capable of inserting the donor comprising a transgene, optionally at a TA dinucleotide site or a TTAA (SEQ ID NO: 440) tetranucleotide site in intron 1, or another intronic site of the CFTR gene. In embodiments, the enzyme associated 24 DB1 / 147031523.8 SAL-017PC / 126933-5017 with the targeting element, is capable of inserting the donor comprising a transgene, optionally a ttTTAAaa (SEQ ID NO: 302) or taTTAAta (SEQ ID NO: 303) octanucleotide site in intron 1, or another intronic site of the CFTR gene. In embodiments, the targeting element directs the helper enzyme to one or more nucleic acid binding sites that are upstream and / or downstream of the TTAA integration sites and within about 5 to about 30 base pairs of the TTAA integration sites or within about 11 to about 15 base pairs of the TTAA integration sites. In embodiments, the targeting element directs the helper enzyme to two nucleic acid binding sites of the TTAA integration sites, wherein a first site is upstream of TTAA and within about 5 to about 30 base pairs or about 11 to about 15 base pairs of the TTAA and a second site is downstream of TTAA and within about 5 to about 30 base pairs or about 11 to about 15 base pairs of the TTAA. In embodiments, the targeting element binds a genomic sequence comprising 20 nucleotides of the 5’ or 3’ end of a TTAA site in intron 1. In embodiments, the TTAA site comprises ttTTAAaa (SEQ ID NO: 302) or taTTAAta (SEQ ID NO: 303) sequences. In embodiments, the targeting elements bind upstream and / or downstream of the TTAA sites. In embodiments, the targeting elements bind within about 10 base pairs, or about 12 base pairs, or about 14 base pairs, or about 16 base pairs, or about 18 base pairs, or about 20 base pairs, or about 22 base pairs or about 24 base pairs, or about 26, base pairs or about 28 base pairs, or about 30 base pairs of the TTAA sites. In embodiments, the binding of a nucleic acid molecule to a targeting element in a mammalian cell is with high target specificity, relative to a control. In embodiments, the control is a composition comprising an enzyme comprising an amino acid sequence of SEQ ID NO: 1 or a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 2 or a codon-optimized form thereof. In embodiments, the targeting element is or comprises one or more of a Cas enzyme, which is optionally catalytically inactive and which is optionally associated with a guide RNA (gRNA), transcription activator-like effector (TALE) DNA binding domain (DBD), Zinc finger, catalytically inactive transcription factor, catalytically inactive nickase, a transcriptional activator, a transcriptional repressor, a recombinase, a DNA methyltransferase, a histone methyltransferase, and a paternally expressed gene 10 (PEG10). In embodiments, the Zinc finger is catalytically inactive. In embodiments, the targeting element is inserted after residue S60, or after residue E70, or after residue L80, or after residue N90, or after residue D100, relative to SEQ ID NO: 1, optionally by way of a linker. In embodiments, the targeting element is inserted immediately after residue G66 relative to SEQ ID NO: 1, optionally by way of a linker. In embodiments, the targeting element is a Zinc finger. In embodiments, the targeting element comprises a 6 finger Zinc finger. In embodiments, the targeting element comprises a 3 finger Zinc finger. In embodiments, the helper enzyme further comprises a zinc finger (E2C) fused to the N-terminus and / or C-terminus. In embodiments, the 25 DB1 / 147031523.8 SAL-017PC / 126933-5017 helper enzyme further comprises a zinc finger (E2C) fused to the N-terminus. In embodiments, the helper enzyme further comprises a zinc finger (E2C) inserted after the glycine at amino acid position 66 relative to SEQ ID NO: 1 within the N-terminus. In embodiments, the helper enzyme is linked to a zinc finger (E2C) using recombinant variable domains of heavy-chain-only antibodies (VHH) (NANOBODY) bound to an ALFA-tag peptide. SEQ ID NO: 304: E2C zinc finger (alone) – nucleotide sequence Ggaggtctcgaaccaggcgagaagccttatgcctgtcctgagtgtggcaaatccttctcaagaaaagactctctggttagacaccagagaacacatacaggg gagaaaccctataaatgccccgaatgcggaaagtccttttcccagagcggcgatctccggaggcatcagagaactcatacaggcgagaaaccatataagtg ccccgagtgtgggaaatccttttccgattgtagagacctggccagacatcaaaggacacatacaggcaagaagaccggaggt SEQ ID NO: 305: E2C zinc finger (alone) – amino acid sequence Gglepgekpyacpecgksfsrkdslvrhqrthtgekpykcpecgksfsqsgdlrrhqrthtgekpykcpecgksfsdcrdlarhqrthtgkktgg In embodiments, the targeting element comprises a Zinc finger. In embodiments, the Zinc finger comprises at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% identity to an amino acid sequence of SEQ ID NO: 305 or a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 304 or a codon-optimized form thereof. In embodiments, the Zinc finger binds a target site in CFTR intron 1. In embodiments, the Zinc finger binds a target site in a genomic safe harbor site (GSHS). In embodiments, the targeting element comprises a TALE DBD. In embodiments, the TALE DBD comprises one or more repeat sequences. In embodiments, the TALE DBD comprises about 14, or about 15, or about, 16, or about 17, or about 18, or about 18.5 repeat sequences. In embodiments, the repeat sequences each independently comprises about 33 or 34 amino acids. In embodiments, the repeat sequences each independently comprises a repeat variable di-residue (RVD) at residue 12 or 13 of the 33 or 34 amino acids, respectively. In embodiments, the RVD recognizes one base pair in a target nucleic acid sequence. In embodiments, the RVD recognizes a C residue in the target nucleic acid sequence and is selected from HD, N(gap), HA, ND, and HI. In embodiments, the RVD recognizes a G residue in the target nucleic acid sequence and is selected from NN, NH, NK, HN, and NA. In embodiments, the RVD recognizes an A residue in the target nucleic acid sequence and is selected from NI and NS. In embodiments, the RVD recognizes a T residue in the target nucleic acid sequence and is selected from NG, HG, H(gap), and IG. In embodiments, the TALE DBD targets one or more introns of the CFTR gene selected from TABLEs 1-4. In embodiments, the TALE DBD is selected from TABLE 4. In embodiments, the TALE DBD binds a target site in CFTR intron 1. In embodiments, the TALE DBD binds a target site in a genomic safe harbor site (GSHS). In embodiments, the targeting element comprises a Cas9 enzyme associated with a gRNA. In embodiments, the Cas9 enzyme associated with a gRNA comprises a catalytically inactive dCas9 associated with a gRNA. 26 DB1 / 147031523.8 SAL-017PC / 126933-5017 In embodiments, the catalytically inactive dCas9 comprises at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% identity to an amino acid sequence of SEQ ID NO: 9 or a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 8 or a codon-optimized form thereof. SEQ ID NO: 8: nucleotide sequence of dead Cas9 DNA binding protein (5004 bp) 1 ATGGACAAGA AGTACTCCAT TGGGCTCGCT ATCGGCACAA ACAGCGTCGG CTGGGCCGTC 61 ATTACGGACG AGTACAAGGT GCCGAGCAAA AAATTCAAAG TTCTGGGCAA TACCGATCGC 121 CACAGCATAA AGAAGAACCT CATTGGCGCC CTCCTGTTCG ACTCCGGGGA GACGGCCGAA 181 GCCACGCGGC TCAAAAGAAC AGCACGGCGC AGATATACCC GCAGAAAGAA TCGGATCTGC 241 TACCTGCAGG AGATCTTTAG TAATGAGATG GCTAAGGTGG ATGACTCTTT CTTCCATAGG 301 CTGGAGGAGT CCTTTTTGGT GGAGGAGGAT AAAAAGCACG AGCGCCACCC AATCTTTGGC 361 AATATCGTGG ACGAGGTGGC GTACCATGAA AAGTACCCAA CCATATATCA TCTGAGGAAG 421 AAGCTTGTAG ACAGTACTGA TAAGGCTGAC TTGCGGTTGA TCTATCTCGC GCTGGCGCAT 481 ATGATCAAAT TTCGGGGACA CTTCCTCATC GAGGGGGACC TGAACCCAGA CAACAGCGAT 541 GTCGACAAAC TCTTTATCCA ACTGGTTCAG ACTTACAATC AGCTTTTCGA AGAGAACCCG 601 ATCAACGCAT CCGGAGTTGA CGCCAAAGCA ATCCTGAGCG CTAGGCTGTC CAAATCCCGG 661 CGGCTCGAAA ACCTCATCGC ACAGCTCCCT GGGGAGAAGA AGAACGGCCT GTTTGGTAAT 721 CTTATCGCCC TGTCACTCGG GCTGACCCCC AACTTTAAAT CTAACTTCGA CCTGGCCGAA 781 GATGCCAAGC TTCAACTGAG CAAAGACACC TACGATGATG ATCTCGACAA TCTGCTGGCC 841 CAGATCGGCG ACCAGTACGC AGACCTTTTT TTGGCGGCAA AGAACCTGTC AGACGCCATT 901 CTGCTGAGTG ATATTCTGCG AGTGAACACG GAGATCACCA AAGCTCCGCT GAGCGCTAGT 961 ATGATCAAGC GCTATGATGA GCACCACCAA GACTTGACTT TGCTGAAGGC CCTTGTCAGA 1021 CAGCAACTGC CTGAGAAGTA CAAGGAAATT TTCTTCGATC AGTCTAAAAA TGGCTACGCC 1081 GGATACATTG ACGGCGGAGC AAGCCAGGAG GAATTTTACA AATTTATTAA GCCCATCTTG 1141 GAAAAAATGG ACGGCACCGA GGAGCTGCTG GTAAAGCTTA ACAGAGAAGA TCTGTTGCGC 1201 AAACAGCGCA CTTTCGACAA TGGAAGCATC CCCCACCAGA TTCACCTGGG CGAACTGCAC 1261 GCTATCCTCA GGCGGCAAGA GGATTTCTAC CCCTTTTTGA AAGATAACAG GGAAAAGATT 1321 GAGAAAATCC TCACATTTCG GATACCCTAC TATGTAGGCC CCCTCGCCCG GGGAAATTCC 1381 AGATTCGCGT GGATGACTCG CAAATCAGAA GAGACCATCA CTCCCTGGAA CTTCGAGGAA 1441 GTCGTGGATA AGGGGGCCTC TGCCCAGTCC TTCATCGAAA GGATGACTAA CTTTGATAAA 1501 AATCTGCCTA ACGAAAAGGT GCTTCCTAAA CACTCTCTGC TGTACGAGTA CTTCACAGTT 1561 TATAACGAGC TCACCAAGGT CAAATACGTC ACAGAAGGGA TGAGAAAGCC AGCATTCCTG 1621 TCTGGAGAGC AGAAGAAAGC TATCGTGGAC CTCCTCTTCA AGACGAACCG GAAAGTTACC 1681 GTGAAACAGC TCAAAGAAGA CTATTTCAAA AAGATTGAAT GTTTCGACTC TGTTGAAATC 1741 AGCGGAGTGG AGGATCGCTT CAACGCATCC CTGGGAACGT ATCACGATCT CCTGAAAATC 1801 ATTAAAGACA AGGACTTCCT GGACAATGAG GAGAACGAGG ACATTCTTGA GGACATTGTC 1861 CTCACCCTTA CGTTGTTTGA AGATAGGGAG ATGATTGAAG AACGCTTGAA AACTTACGCT 1921 CATCTCTTCG ACGACAAAGT CATGAAACAG CTCAAGAGGC GCCGATATAC AGGATGGGGG 1981 CGGCTGTCAA GAAAACTGAT CAATGGGATC CGAGACAAGC AGAGTGGAAA GACAATCCTG 2041 GATTTTCTTA AGTCCGATGG ATTTGCCAAC CGGAACTTCA TGCAGTTGAT CCATGATGAC 2101 TCTCTCACCT TTAAGGAGGA CATCCAGAAA GCACAAGTTT CTGGCCAGGG GGACAGTCTT 2161 CACGAGCACA TCGCTAATCT TGCAGGTAGC CCAGCTATCA AAAAGGGAAT ACTGCAGACC 2221 GTTAAGGTCG TGGATGAACT CGTCAAAGTA ATGGGAAGGC ATAAGCCCGA GAATATCGTT 2281 ATCGAGATGG CCCGAGAGAA CCAAACTACC CAGAAGGGAC AGAAGAACAG TAGGGAAAGG 2341 ATGAAGAGGA TTGAAGAGGG TATAAAAGAA CTGGGGTCCC AAATCCTTAA GGAACACCCA 2401 GTTGAAAACA CCCAGCTTCA GAATGAGAAG CTCTACCTGT ACTACCTGCA GAACGGCAGG 2461 GACATGTACG TGGATCAGGA ACTGGACATC AATCGGCTCT CCGACTACGA CGTGGCTGCT 2521 ATCGTGCCCC AGTCTTTTCT CAAAGATGAT TCTATTGATA ATAAAGTGTT GACAAGATCC 2581 GATAAAGCTA GAGGGAAGAG TGATAACGTC CCCTCAGAAG AAGTTGTCAA GAAAATGAAA 2641 AATTATTGGC GGCAGCTGCT GAACGCCAAA CTGATCACAC AACGGAAGTT CGATAATCTG 2701 ACTAAGGCTG AACGAGGTGG CCTGTCTGAG TTGGATAAAG CCGGCTTCAT CAAAAGGCAG 2761 CTTGTTGAGA CACGCCAGAT CACCAAGCAC GTGGCCCAAA TTCTCGATTC ACGCATGAAC 2821 ACCAAGTACG ATGAAAATGA CAAACTGATT CGAGAGGTGA AAGTTATTAC TCTGAAGTCT 2881 AAGCTGGTCT CAGATTTCAG AAAGGACTTT CAGTTTTATA AGGTGAGAGA GATCAACAAT 2941 TACCACCATG CGCATGATGC CTACCTGAAT GCAGTGGTAG GCACTGCACT TATCAAAAAA 3001 TATCCCAAGC TTGAATCTGA ATTTGTTTAC GGAGACTATA AAGTGTACGA TGTTAGGAAA 27 DB1 / 147031523.8 SAL-017PC / 126933-5017 3061 ATGATCGCAA AGTCTGAGCA GGAAATAGGC AAGGCCACCG CTAAGTACTT CTTTTACAGC 3121 AATATTATGA ATTTTTTCAA GACCGAGATT ACACTGGCCA ATGGAGAGAT TCGGAAGCGA 3181 CCACTTATCG AAACAAACGG AGAAACAGGA GAAATCGTGT GGGACAAGGG TAGGGATTTC 3241 GCGACAGTCC GGAAGGTCCT GTCCATGCCG CAGGTGAACA TCGTTAAAAA GACCGAAGTA 3301 CAGACCGGAG GCTTCTCCAA GGAAAGTATC CTCCCGAAAA GGAACAGCGA CAAGCTGATC 3361 GCACGCAAAA AAGATTGGGA CCCCAAGAAA TACGGCGGAT TCGATTCTCC TACAGTCGCT 3421 TACAGTGTAC TGGTTGTGGC CAAAGTGGAG AAAGGGAAGT CTAAAAAACT CAAAAGCGTC 3481 AAGGAACTGC TGGGCATCAC AATCATGGAG CGATCAAGCT TCGAAAAAAA CCCCATCGAC 3541 TTTCTGGAGG CGAAAGGATA TAAAGAGGTC AAAAAAGACC TCATCATTAA GCTTCCCAAG 3601 TACTCTCTCT TTGAGCTTGA AAACGGCCGG AAACGAATGC TCGCTAGTGC GGGCGAGCTG 3661 CAGAAAGGTA ACGAGCTGGC ACTGCCCTCT AAATACGTTA ATTTCTTGTA TCTGGCCAGC 3721 CACTATGAAA AGCTCAAAGG GTCTCCCGAA GATAATGAGC AGAAGCAGCT GTTCGTGGAA 3781 CAACACAAAC ACTACCTTGA TGAGATCATC GAGCAAATAA GCGAATTCTC CAAAAGAGTG 3841 ATCCTCGCCG ACGCTAACCT CGATAAGGTG CTTTCTGCTT ACAATAAGCA CAGGGATAAG 3901 CCCATCAGGG AGCAGGCAGA AAACATTATC CACTTGTTTA CTCTGACCAA CTTGGGCGCG 3961 CCTGCAGCCT TCAAGTACTT CGACACCACC ATAGACAGAA AGCGGTACAC CTCTACAAAG 4021 GAGGTCCTGG ACGCCACACT GATTCATCAG TCAATTACGG GGCTCTATGA AACAAGAATC 4081 GACCTCTCTC AGCTCGGTGG AGAC SEQ ID NO: 9: amino acid sequence of dead Cas9 DNA binding protein (1368 amino acids) 1 MDKKYSIGLA IGTNSVGWAV ITDEYKVPSK KFKVLGNTDR HSIKKNLIGA LLFDSGETAE 61 ATRLKRTARR RYTRRKNRIC YLQEIFSNEM AKVDDSFFHR LEESFLVEED KKHERHPIFG 121 NIVDEVAYHE KYPTIYHLRK KLVDSTDKAD LRLIYLALAH MIKFRGHFLI EGDLNPDNSD 181 VDKLFIQLVQ TYNQLFEENP INASGVDAKA ILSARLSKSR RLENLIAQLP GEKKNGLFGN 241 LIALSLGLTP NFKSNFDLAE DAKLQLSKDT YDDDLDNLLA QIGDQYADLF LAAKNLSDAI 301 LLSDILRVNT EITKAPLSAS MIKRYDEHHQ DLTLLKALVR QQLPEKYKEI FFDQSKNGYA 361 GYIDGGASQE EFYKFIKPIL EKMDGTEELL VKLNREDLLR KQRTFDNGSI PHQIHLGELH 421 AILRRQEDFY PFLKDNREKI EKILTFRIPY YVGPLARGNS RFAWMTRKSE ETITPWNFEE 481 VVDKGASAQS FIERMTNFDK NLPNEKVLPK HSLLYEYFTV YNELTKVKYV TEGMRKPAFL 541 SGEQKKAIVD LLFKTNRKVT VKQLKEDYFK KIECFDSVEI SGVEDRFNAS LGTYHDLLKI 601 IKDKDFLDNE ENEDILEDIV LTLTLFEDRE MIEERLKTYA HLFDDKVMKQ LKRRRYTGWG 661 RLSRKLINGI RDKQSGKTIL DFLKSDGFAN RNFMQLIHDD SLTFKEDIQK AQVSGQGDSL 721 HEHIANLAGS PAIKKGILQT VKVVDELVKV MGRHKPENIV IEMARENQTT QKGQKNSRER 781 MKRIEEGIKE LGSQILKEHP VENTQLQNEK LYLYYLQNGR DMYVDQELDI NRLSDYDVAA 841 IVPQSFLKDD SIDNKVLTRS DKARGKSDNV PSEEVVKKMK NYWRQLLNAK LITQRKFDNL 901 TKAERGGLSE LDKAGFIKRQ LVETRQITKH VAQILDSRMN TKYDENDKLI REVKVITLKS 961 KLVSDFRKDF QFYKVREINN YHHAHDAYLN AVVGTALIKK YPKLESEFVY GDYKVYDVRK 1021 MIAKSEQEIG KATAKYFFYS NIMNFFKTEI TLANGEIRKR PLIETNGETG EIVWDKGRDF 1081 ATVRKVLSMP QVNIVKKTEV QTGGFSKESI LPKRNSDKLI ARKKDWDPKK YGGFDSPTVA 1141 YSVLVVAKVE KGKSKKLKSV KELLGITIME RSSFEKNPID FLEAKGYKEV KKDLIIKLPK 1201 YSLFELENGR KRMLASAGEL QKGNELALPS KYVNFLYLAS HYEKLKGSPE DNEQKQLFVE 1261 QHKHYLDEII EQISEFSKRV ILADANLDKV LSAYNKHRDK PIREQAENII HLFTLTNLGA 1321 PAAFKYFDTT IDRKRYTSTK EVLDATLIHQ SITGLYETRI DLSQLGGD In embodiments, the targeting element comprises a Cas12 enzyme associated with a gRNA. In embodiments, the targeting element comprises a catalytically inactive Cas12 associated with a gRNA, optionally wherein the catalytically inactive Cas12 is dCas12j or dCas12a. In embodiments, the targeting element comprises a Cas6, Cas7, or Cas8 enzyme associated with a gRNA. In embodiments, the target element binds a target site selected from any one of the targets sites of Tables 1-4. In embodiments, the targeting elements binds a genomic sequence comprising 20 nucleotides of the 5’ or 3’ end of a TTAA site in intron 1, or a variant thereof comprising substitutions or deletions. In embodiments, the TTAA site comprises ttTTAAaa (SEQ ID NO: 302) or taTTAAta (SEQ ID NO: 303) sequences. 28 DB1 / 147031523.8 SAL-017PC / 126933-5017 Guide RNA sequences targeting the CFTR intron site are listed in TABLEs 1-4. 29 DB1 / 147031523.87105 S- FT33S HSTSFTS FHKRSFTST FTSHSFTST TKR S HTK S TRGQ S HKR SHT KTRS HKGR9K6G2CRGQ KQCEHRGRG KQCEGCQCHHEPAKTRGRGQCQ KR GEH GCQCEQKTHGRQCQEHA1E HP ECE HP E R CLC / P AE HPVNEHP NCE HPRCL P ARC P RLKNAP E CL PNKCKLP VCRKLCCPK7YLKHNYPACRKYLK CNYPKYDYGPKRCTQKLKNYE CL KPDRKYNYPNCNDKLEYNPAR1P A0RKQ P ARK P S E SYP GKR PD-KKKYRP DRKEQLAS :7rhc,ATAAT GAG81F G AAGG3A Tgh(1 nA Aor2 TG AAA AtFGT GTA GTAAAniRTF3AAA ACAAGAGTCF AGT G C A AnisregAni4G A GFACAAATGATGAGCG FcniZA G Gr5FAGAA G ACA A T AegA T A GniFxiA A A G A8.S6.FGGAGAGATAAAAA32151E3L 1 5 407B0ADI53Z50Z514Z24Z40 4Z5Z1T / 1BD7105 S- F33STSSHKFTS SHT FTSTSFTST FHKTRSFTS FHKTRFH TKR SKHT KR S HTKR S T GQ S HTK S TRGQ SKT9KT G K GQ GQ K GQ KR CH K G K CH R621 / CP710-LAS ATCAG A G AG GGTAGTAGTTGGGTA A A T A AAAGACACAACGACTAA A G TAAAGAGAAATAAT G G GGAAAG T A A A GGAATGTGGATACAAAAAAG A A G TAAA AGAAAAAATGAAAAAAGGAA A C G ACAAAACAATCA8.CAC325138070 7 2 2 8 8 9 758 5 2 1 1 9 9 4Z5Z6Z9Z2Z2Z4Z4Z1 / 1BD7105- KR FTHSTS33G9CQHSKTRKGRFTS QSKHTKT FTGRQSKHS TKTSFG GRQSKTFH FHKTRHSKTRSKTRGCQH621 / CP710-LAS AAT TAACGGGAT GTGT AAAATGAG TTG AAAACGAAAAAGAAAAG AAAAAGAA AAG AAAAAAAGCTCGACAATCAAA A GG GTGACTAAATAGTA GATAATCGG T G AGTATGATTGAGA AACGAAAA A GAAAAG A G AAAAAAAAAAA A A C8AGAAAGCTGA.C325130007574 2 9 74Z55Z60Z91Z9Z1 / 1BD7105-C C3E3PEPCE CECECECPEPECPEC CE C E PEPEP P E P9C C PP6K2YKYCK C CPK KC CKKC CYKYKCKCKCYKPYCCKKY1 / K K YY YKYK KYKYC E E KK KE EK KEKEYKKEP710-LASg h( ONO:O:O:O:OONO:NO O:NONO:NO ONA D N N N N ND D ND NnIDIDIigQDIDIDI I IDIDIDI IQ Q QDIDIo Q Q Q Q Q Q Q Q Q QeESER (SE(S( ESESE E(S(SE(S( ESESE E(S(S( ESES(1neG T T(A G A G T(C G A G(tATGATGT A G T GGA T G AT ACA AAoirStnt AGTAAC C TTTTAGTGTAAGCTTC TACACCCCAATGAi egRrGTG A G T C A G A C A AAG G A TTACCTA A T C AA ACATACT aT CTAGTTA C C GAACAAATA G G GFCnisT TrA G A G T C G A G T Ae1GgFATGATGTCACGTTGGGACTCGTACACAAniFcniZ 2A TrFGGAAGTTATA TGGTTACAGATT CGAAACCCC A GAAATAAAegniFeer3T G T A C A A T A A AhFAG ACTAGTTATCCCTGAACAACATACGTGCG8.T3.22E18283 4 8 9 0 4 9 3 5 2 3 4 513L 6BZ68Z68Z69Z69Z64Z74 5 6 6 8 8 4 0Z7Z7Z7Z7Z7Z7 8 7D F F F F F F F F F F F FZFZF41AI C C C C C C C C C C C C C CT / 1BD7105-C3ECECECEE3P P P P PECPECPECPECPECCP EECEECECE9PP P P P P621 / CP710-LAS DIDIDIDIDIDIDID D D D D DQEQ Q QDI I I IDIQ Q Q QDSE(S SEA( (SET(A(A(SEG(A S(SE(SE T(SEG(ST(A S(S(ST(S(AGC A G A CTT G G TATCTAAAG G A C ATA C A G G AGGAAAA G A C A A T G T A G TATAGACAAAACAAAAGGAAATGAATAATCCCTGTAATCATCTA A GACAGAAAGGGGGATAGCAG A A T T G TAAAGAAGTGTATGTAAAG TAAACAGAA G A T T G TGGAACCATATCGAGAGTA T C AGTAGATGAAAGA A A A TACG CAAACAAAAT GGGAAATTGAAATGAATCCCTTGTAATCAATTACAGGAAGG AAG G A AATTT GTTC A A A G GGT CAAAAGA G GTA GTA8.3251 2 2 3 4 514 5 5 7 7 75 9 8 8 1 3 0 0 0 7 38 8 8 8 8 87 1 2 5 6 6 0 0 0 0 0Z8 9 9Z9 9 9 1Z1Z1Z1Z7FZFZFZFZFZFZ Z Z Z Z 4C C C C C CFCFCFCFCFCFCFC 6FC 7FC 8FC 21 / 1BD7105-C3ECECC ECEC CKCKK K3P PEP E9PPCPEP Y Y YPYP621 / CP710-LAS CED CSPS SCARCD CN CA CG E S E S E FSE FSCREP QEP QEPKREPQSE R E A P F P F PKSYFCKSFCKSFCKSFCKFP Q P Q CS CS CKPCKSCKSFCKSFKYKGKKGKYGCKGCKSEKYSGKEKYSGKEKYGKSEKYGKKEGYCKSEKYGKSEKP CYPGKEEPKC PEEPKEYEPP ECKEPC53)6) ) ) ) ) )87 5) )8)1888980)91 29 270 090:1:1:19:1 1 1:2:3:3:3:OO O O: :ONO O ONN NONO ON N N ND N NIDIDIDIDIDIDIDIDIDID IQEQ Q Q Q Q QQEQ QEQE SE(SE(SE(SE(SE E(S(S( S( ES(S(S(GTA A G A A A A A AGTAGAAA TACA GTAATGAAGTAT AAAGAATT AAGC AA TTGAA GAAATA TAAAGATTCACGAT CAAGCAAGAAAGTTCCCAAGAAAGACGTATA G A A A A ATA AG GAAAATCAAGTAA GAAGTAATAGAATTGCAAAAAGTTTGCAACGGTAACAAATA T A T A A A GCAAGAAAAGTTCCCAAGAAAGAC8.32051111112 2 2 2 31 1 111111 1 0Z Z Z Z Z1 1 A B C 7FC 9FC 0FC 1FC 0FZC 1FZC 2F0C 350Z50Z50Z54Z1 / 1BD7105- S S33F FS S S S STSFSFSFSFSFS SS FFS S S S FF F FS SF9S6K2GKGK K K K KS SKSKSKSK KSK1C CGCGCG G GKCG G G G G GCG / E E E ECECE EC CEC CECE ECC E EPEP710-LASg h: : : : :(:O:O:OO O:O:O: :O O:O:OOBN N N N N NO ON N ND N N N D N NnoDIDIDI IDIDID D DIDIDIDIDIigQ Q Q Q Q I I IQ Q Q Q Q Q Q Q QeERSE(SE E(S(SE(SE(SE(SE E E(S S(S( ESE E E(S(S(S(1neA T T G AtoiT T G AAAT(AAG T A ACA T C CAGAT T TAAAAACArt SAnit TGAAA A A CTTGTAT A A A GeT A A AAAAT T AGA A C A A GRgrGAAT a AAAAAAAAAG AGAACA T CAATTTAGGTC C TAGTG AF T A A A G C A A C A A C ATAAAC nisre A TgTT G A T G TTA ACA T G Tn1iFTA GGAAAAAACAT GCA GAAAAGTCAFcniZT A A A C T A A A Gr2 T A A AAA TTTTAGA A C A A GeF G A A A A G A A T C G C A TgniFAAA A AAAGA C ATT A TAGTACAe3F AAAAA G CAAAA CTAGA CAer A G A8h.T3.3 32425262720173830 241434445484 51E 3 3 3 3 3 4 4 4 43L 4BZ4FZ4FZ4FZ44 4 4 4 4 0FZ4FZ4FZ4FZ4FZ4FZ4FZ4 4 4 7FZFZFZF41A DI C C C C C C C C C C C C C CT / 1BD7105-T3SQ3FSSFSFS STFSSFSRFSSF TSS RS RSSF9SFS SFS SFS S SFSF FS FFS621 / CP710-LAS DID NIDIDIDQDI IDIDIDIDIDIDIDIDID DQQ Q I IQEQSE ES QE ESEQ Q Q Q QSE ESE E EQSESEQSEQ QSE E(S(S( (S(S(S( ( ( ( (S SA(CA G(A A(T T C C T C G( (ACATAAA GT C A T A AGT A C C AGA GGG GC G C C ACG A G C G G T AAT AT TACTAT AGGA CTAAAG AG T A G A A A ACG GTCAAA A G TGC G ATAG AGA AA T ATT G AT GTTTG AA AATAA C C A A GAA C AGTTATA G A AAAACA G G A A T T C C T C G G ACAAAATCCGACTCAAAGGTAAGCCCGAGATGATGG A C GATG T TCAATAAGCTAGGAGGTT ACAAACAAGGA A AATAGAAAAATTGATTAGCATAGTTTG GTA TAGAATAA C C A A G A C AGT ATA G A AAA8.315455567102090728284274 25556 7 0514 4 4 4 5 5 5 5 5 5 5 5 555555 34 4 4 4 4 4 4 4 4 4 4 4 4 4 4460ZFZ Z Z Z Z Z Z Z Z Z Z Z Z Z Z74CFCFCFCFCFCFCFCFCFCFCFCFCFCFCFC1 / 1BD7105- S P P33F C C9S K K621 / CP710-LAS R QNHANDVR STQ RLSNFSGSFSTK KTG GSC CFE ESP PKC)GK9CK)0CYEP22Y3PK:P 2:CEKGOKNEGONYTHDITHDT I KETQER QGRT )QHS( QEHS(HT60R S R SR 1Q: VLTKR THOKL KRN NDGDKVD S TGGTL IR HSHQ TQHTGESFPS SRSQFSRQD(SGKGHAKHAFTSHCERGLCRLKTGRPCNEAPCNACQEHKYRQKYRQ8PRVPKSFPKSF3CKLYNE S E SKSGK GKESP G P GQELCEELCE)3) )272282:2:2:OO ONN NDIDIDI QQE QE E S( S(S(AAG AGA CTGAGAGAA ACAAGGGAAAAAAAGGAAGCGTAA GAAAAGCGAAAG AAA8.3821511310Z8F94 74C 740Z5Z1 / 1BD SAL-017PC / 126933-5017 TABLE 4. TALES in CFTR intron 1 (hg38, chr7:117488023-117489022) used to target TTAA Sites Name Target Site RVD L-TALE1-IN TGCCTCTTTTTTTGG(SEQ ID NO: NG-NN-HD-HD-NG-HD-NG-NG-NG-NG- 107) NG-NG-NG-NN-NN(SEQ ID NO: 120) - : - : - : - : : 9) D - - - DB1 / 147031523.8 SAL-017PC / 126933-5017 RA-TALE12-19-R AATATTTTGGAG (SEQ ID NO: NI-NI-NG-NI-NG-NG-NG-NG-NN-NN-NI- 238) NN (SEQ ID NO: 273) RA-TALE9-19-R ATTTTGGAG (SEQ ID NO: 239) NI-NG-NG-NG-NG-NN-NN-NI-NG (SEQ I- - - - Q I- D D - - - I- 40 DB1 / 147031523.8 SAL-017PC / 126933-5017 RH-TALE9-19-R TTTAGAGAA (SEQ ID NO: 263) NG-NG-NG-NI-NN-NI-NN-NI-NI (SEQ ID NO: 298) RH-TALE13-13-R TAGAGAACAGAAG (SEQ ID NO: NG-NI-NN-NI-NN-NI-NI-HD-NI-NN-NI- N t 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 mutations. In embodiments, the guide RNA targets one or more sites selected from TABLEs 1-4. In embodiments, the zinc finger comprises one of the sequences selected from TABLEs 1-4, or variants thereof comprising about 99, about 98, about 97, about 95, about 94, about 93, about 92, about 91, about 90, about 89, about 88, about 87, about 86, about 85, about 84, about 83, about 82, about 81, about 80 percent identity to the sequence. In embodiments, the zinc finger targets one or more sites selected from TABLEs 1-4. In embodiments, the targeting element comprises a Cas9 enzyme guide RNA complex. In embodiments, the Cas9 enzyme guide RNA complex comprises a nuclease-deficient dCas9 guide RNA complex. In embodiments, the targeting element comprises a Cas12 enzyme guide RNA complex. In embodiments, the targeting element comprises a nuclease-deficient dCas12 guide RNA complex, optionally dCas12j guide RNA complex or dCas12a guide RNA complex. In embodiments, the targeting element comprises a Cas12k enzyme guide RNA complex. In embodiments, the targeting element comprises a nuclease-deficient dCas12 guide RNA complex, optionally dCas12k guide RNA complex. In embodiments, a targeting chimeric system or construct, having a DBD fused to the helper enzyme directs binding of the helper to a specific sequence (e.g., transcription activator-like effector proteins (TALE) repeat variable di- residues (RVD) or gRNA) near an enzyme recognition site. The enzyme is thus prevented from binding to random recognition sites. In embodiments, the targeting chimeric construct binds to a human CFTR intron 1 or other CFTR introns. In embodiments, the helper enzyme is capable of targeted genomic integration by transposition is linked to or fused with a TALE DNA binding domain (DBD) or a Cas-based gene-editing system, such as, e.g., dCasX, Cas9 or a variant thereof. In embodiments, the targeting element targets the helper enzyme to a locus of interest. In embodiments, the targeting element comprises CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat) associated protein 9 (Cas9), or a variant thereof. A CRISPR / Cas9 tool only requires Cas9 nuclease for DNA cleavage and a single-guide RNA (sgRNA) for target specificity. See Jinek et al. (2012) Science 337, 816–821; Chylinski et al. (2014) Nucleic Acids Res 42, 6091–6105. The inactivated form of Cas9, which is a nuclease-deficient (or inactive, 41 DB1 / 147031523.8 SAL-017PC / 126933-5017 or “catalytically dead” Cas9, is typically denoted as “dCas9,” has no substantial nuclease activity. Qi, L. S. et al. (2013). Cell 152, 1173–1183. CRISPR / dCas9 binds precisely to specific genomic sequences through targeting of guide RNA (gRNA) sequences. See Dominguez et al., Nat Rev Mol Cell Biol.2016;17:5–15; Wang et al., Annu Rev Biochem.2016;85:227–64. dCas9 is utilized to edit gene expression when applied to the transcription binding site of a desired site and / or locus in a genome. When the dCas9 protein is coupled to guide RNA (gRNA) to create dCas9 guide RNA complex, dCas9 prevents the proliferation of repeating codons and DNA sequences that might be harmful to an organism's genome. Essentially, when multiple repeat codons are produced, it elicits a response, or recruits an abundance of dCas9 to combat the overproduction of those codons and results in the shut-down of transcription. Thus, dCas9 works synergistically with gRNA and directly affects the DNA polymerase II from continuing transcription. In embodiments, the targeting element comprises a nuclease-deficient Cas enzyme guide RNA complex. In embodiments, the targeting element comprises a nuclease-deficient (or inactive, or “catalytically dead” Cas, e.g., Cas9, typically denoted as “dCas” or “dCas9”) guide RNA complex. In embodiments, a Cas-based targeting element comprises Cas12 or a variant thereof, e.g., without limitation, Cas12a (e.g., dCas12a), or Cas12j (e.g., dCas12j), or Cas12k (e.g., dCas12k). In embodiments, the targeting element comprises a Cas12 enzyme guide RNA complex. In embodiments, comprises a nuclease-deficient dCas12 guide RNA complex, optionally dCas12j guide RNA complex or dCas12a guide RNA complex. In embodiments, the targeting element is selected from a zinc finger (ZF), transcription activator-like effector (TALE), meganuclease, and clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein, any of which are, in embodiments, catalytically inactive. In embodiments, the CRISPR-associated protein is selected from Cas9, CasX, CasY, Cas12a (Cpf1), and gRNA complexes thereof. In embodiments, the CRISPR- associated protein is selected from Cas9, xCas9, Cas 6, Cas7, Cas8, Cas12a (Cpf1), Cas13a, Cas14, CasX, CasY, a Class 1 Cas protein, a Class 2 Cas protein, MAD7, MG1 nuclease, MG2 nuclease, MG3 nuclease, or catalytically inactive forms thereof, and gRNA complexes thereof. In embodiments, the helper enzyme of the present disclosure is capable of inserting a donor DNA at a TA dinucleotide site or a TTAA tetranucleotide site in an intron of a gene, e.g., CFTR. The helper enzyme of the present disclosure is suitable for causing insertion of the donor DNA in an intron when contacted with a biological cell. In embodiments, the helper enzyme of the present disclosure is capable of inserting a donor DNA at a ttTATAaa (SEQ ID NO: 302) or taTTAAta (SEQ ID NO: 303) octanucleotide site in an intron of a gene, e.g., CFTR. The helper enzyme of the present disclosure is suitable for causing insertion of the donor DNA in an intron when contacted with a biological cell. In embodiments, the targeting element is suitable for directing the helper enzyme of the present disclosure to the CFTR intron sequence. 42 DB1 / 147031523.8 SAL-017PC / 126933-5017 In embodiments, the targeting element comprises transcription activator-like effector (TALE) DNA binding domain (DBD). The TALE DBD comprises one or more repeat sequences. For example, in embodiments, the TALE DBD comprises about 14, or about 15, or about, 16, or about 17, or about 18, or about 18.5 repeat sequences. In embodiments, the TALE DBD repeat sequences comprise 33 or 34 amino acids. In embodiments, the one or more of the TALE DBD repeat sequences comprise a repeat variable di-residue (RVD) at residue 12 or 13 of the 33 or 34 amino acids. In embodiments, the targeting element (e.g., TALE or Zinc Fingers) DBDs cause the helper enzyme of the present disclosure to bind specifically to human CFTR intron 1 or other CFTR introns. In embodiments, the TALEs or Zinc Finger DBDs sequester the helper to CFTR introns (e.g., intron 1) and promote transposition to nearby TA dinucleotide or a TTAA tetranucleotide sites, or a ttTTAAaa (SEQ ID NO: 302) or taTTAAta (SEQ ID NO: 303) octanucleotide site which can be located in proximity to the repeat variable di-residues (RVD) TALE or Zinc FInger nucleotide sequences. Accordingly, the helper enzyme of the present disclosure does not only operate based on its ability to recognize TA, TTAA, ttTTAAaa (SEQ ID NO: 302), or taTTAAta (SEQ ID NO: 303) sites, but it also directs a donor DNA (having a transgene) to specific locations in proximity to a TALE or Zinc Finger DBD. In embodiments, the targeting element directs the helper enzyme to either one or more nucleic acid binding sites that are upstream and / or downstream of the TTAA integration sites or to the TTAA integration sites. In embodiments, the targeting element directs the helper enzyme to one or more nucleic acid binding sites that are upstream and / or downstream of the TTAA integration sites and within about 5 to about 30 base pairs of the TTAA integration sites or within about 11 to about 15 base pairs of the TTAA integration sites. In embodiments, the targeting element directs the helper enzyme to two nucleic acid binding sites of the TTAA integration sites, wherein a first site is upstream of TTAA and within about 5 to about 30 base pairs or about 11 to about 15 base pairs of the TTAA and a second site is downstream of TTAA and within about 5 to about 30 base pairs or about 11 to about 15 base pairs of the TTAA. The helper enzyme of the present disclosure in accordance with embodiments of the present disclosure has negligible risk of genotoxicity and exhibits superior features as compared to existing gene therapies. In embodiments, the helper enzyme of the present disclosure is mutated to be characterized by reduced or inhibited binding of off-target sequences and consequently reliant on a DBD fused thereto, such as a TALE or Zinc Finger DBD, for transposition. The described cells, compositions, and methods allow reducing vector and transgene insertions that increase a mutagenic risk. The described cells and methods make use of a gene transfer system that reduces genotoxicity compared to viral- and nuclease-mediated gene therapies. In embodiments, TALE or Zinc Finger DBDs are customizable, such as a TALE or Zinc Finger DBDs is selected for targeting a specific genomic location. In embodiments, the genomic location is in proximity to a TA dinucleotide 43 DB1 / 147031523.8 SAL-017PC / 126933-5017 site or a TTAA (SEQ ID NO: 440) tetranucleotide site, or a ttTTAAaa (SEQ ID NO: 302) or a taTTAAta (SEQ ID NO: 303) octanucleotide site. Embodiments of the present disclosure make use of the ability of TALE or Zinc Finger DBDs to target specific sites in a host genome. The DNA targeting ability of a TALE or Zinc Finger DBD is provided by TALE repeat sequences (e.g., modular arrays) or Zinc Finger which are linked together to recognize flanking DNA sequences. Each TALE or Zinc Finger can recognize certain base pair(s) or residue(s). In embodiments, the Zinc finger binding site is different in the donor and the target site. In embodiments, the Zinc finger binding site is the same in the donor and the target site. TALE nucleases (TALENs) are a known tool for genome editing and introducing targeted double-stranded breaks. TALENs comprise endonucleases, such as FokI nuclease domain, fused to a customizable DBD. This DBD is composed of highly conserved repeats from TALEs, which are proteins secreted by Xanthomonas bacteria to alter transcription of genes in host plant cells. The DBD includes a repeated highly conserved 33–34 amino acid sequence with divergent 12th and 13th amino acids. These two positions, referred to as the RVD, are highly variable and show a strong correlation with specific base pair or nucleotide recognition. This straightforward relationship between amino acid sequence and DNA recognition has allowed for the engineering of specific DBDs by selecting a combination of repeat segments containing the appropriate RVDs. Boch et al. Nat. Biotechnol.2011; 29 (2): 135–6. Accordingly, TALENs can be readily designed using a “protein-DNA code” that relates modular DNA-binding TALE repeat domains to individual bases in a target-binding site. See Joung et al. Nat Rev Mol Cell Biol.2013;14(1):49- 55. The following table, for example, shows such code: RVD Nucleotide RVD Nucleotide It has been demonstrated that TALENs can be used to target essentially any DNA sequence of interest in human cell. Miller et al. Nat. Biotechnol.2011;29:143–148. Guidelines for selection of potential target sites and for use of particular TALE repeat domains (harboring NH residues at the hypervariable positions) for recognition of G bases have been proposed. See Streubel et al. Nat. Biotechnol.2012;30:593–595. 44 DB1 / 147031523.8 SAL-017PC / 126933-5017 Accordingly, in embodiments, the TALE DBD comprises one or more repeat sequences. In embodiments, the TALE DBD comprises about 15, or about, 16, or about 17, or about 18, or about 18.5 repeat sequences. In embodiments, the TALE DBD repeat sequences comprise 33 or 34 amino acids. In embodiments, the one or more of the TALE DBD repeat sequences comprise an RVD at residue 12 or 13 of the 33 or 34 amino acids. The RVD can recognize certain base pair(s) or residue(s). In embodiments, the RVD recognizes one base pair in the nucleic acid molecule. In embodiments, the RVD recognizes a C residue in the nucleic acid molecule and is selected from HD, N(gap), HA, ND, and HI. In embodiments, the RVD recognizes a G residue in the nucleic acid molecule and is selected from NN, NH, NK, HN, and NA. In embodiments, the RVD recognizes an A residue in the nucleic acid molecule and is selected from NI and NS. In embodiments, the RVD recognizes a T residue in the nucleic acid molecule and is selected from NG, HG, H(gap), and IG. In embodiments, the present disclosure relates to a system having nucleic acids encoding the enzyme (e.g., without limitation, the helper enzyme) and the donor DNA, respectively. In embodiments, a nucleic acid encoding the enzyme (e.g., without limitation, the helper enzyme) is RNA. In embodiments, a nucleic acid encoding the transgene is DNA. In embodiments, thr RNA is or comprises messenger RNA (mRNA). In embodiments, the mRNA is or comprises modified mRNA (mmRNA). In embodiments, the enzyme (e.g., without limitation, the helper enzyme) is encoded by a recombinant or synthetic nucleic acid. In embodiments, the nucleic acid is RNA, optionally a helper RNA. In embodiments, the nucleic acid is RNA that has a 5’-m7G cap (cap0, or cap1, or cap2), optionally with pseudouridine substitution (e.g., without limitation n-methyl-pseudouridine), and optionally a poly-A tail of about 30, or about 50, or about 100, of about 150 nucleotides in length. In embodiments, the poly-A tail is of about 30 nucleotides in length, optionally 34 nucleotides in length. In embodiments, a nuclear localization signal is placed before the enzyme start codon at the N-terminus, optionally at the C-terminus. In embodiments, the nucleic acid that is RNA has a 5’-m7G cap (cap 0, or cap 1, or cap 2). In embodiments, the nucleic acid comprises a 5’ cap structure, a 5’-UTR comprising a Kozak consensus sequence, a 5′-UTR comprising a sequence that increases RNA stability in vivo, a 3’-UTR comprising a sequence that increases RNA stability in vivo, and / or a 3’ poly(A) tail. In embodiments, the enzyme (e.g., without limitation, a helper) is incorporated into a vector or a vector-like particle. In embodiments, the vector is a non-viral vector. In embodiments, a nucleic acid encoding the helper enzyme in accordance with embodiments of the present disclosure, is DNA. In various embodiments, a construct comprising a donor is any suitable genetic construct, such as a nucleic acid construct, a plasmid, or a vector. In various embodiments, the construct is DNA, which is referred to herein as a 45 DB1 / 147031523.8 SAL-017PC / 126933-5017 donor DNA. In embodiments, sequences of a nucleic acid encoding the donor is codon optimized to provide improved mRNA stability and protein expression in mammalian systems. In embodiments, the helper enzyme and the donor are included in different vectors. In embodiments, the helper enzyme and the donor are included in the same vector. In various embodiments, a nucleic acid encoding the helper enzyme capable of targeted genomic integration by transposition (e.g., without limitation, the helper enzyme) is RNA (e.g., helper RNA), and a nucleic acid encoding a donor is DNA. In embodiments, the one or more nucleic acid-based agents is in the form of DNA. In embodiments, the DNA is or comprises plasmid DNA. In embodiments, the plasmid DNA has a size of up to about 10 kb, or up to about 12 kb, up to about 15 kb, or about 7.5 to about 15 kb, or about 10 to about 15 kb. In embodiments, the plasmid is conjugative, non-conjugative or mobilized. In embodiments, the plasmid is selected from fertility F-plasmids, resistance plasmids, Col plasmids, degradative plasmids, or virulence plasmids. In embodiments, the plasmid is an artificially construct plasmid that is used as vectors in genetic engineering. In embodiments, the plasmid is selected from miniplasmid, nanoplasmid, doggybone or close-ended linear DNA. In embodiments, the composition further comprises a donor nucleic acid or is suitable for insertion of a donor nucleic acid, optionally wherein the donor nucleic acid is a transposon. In embodiments, the helper enzyme is suitable of inserting a donor nucleic acid comprising a transgene in a genomic safe harbor site (GSHS) and / or wherein the targeting element is suitable for directing the helper enzyme to a GSHS. In embodiments, a donor DNA and a helper RNA are suitable for transfection at a donor DNA to helper RNA ratio of about 1 to about 4, or about 1 to about 2, or about 1 to about 1. In embodiments, the composition further comprises a nucleic acid encoding a donor comprising a transgene to be integrated, optionally wherein the transgene is defective or substantially absent in a disease state. In embodiments, the transgene comprises a cargo nucleic acid sequence and a first and a second donor end sequences. In embodiments, transgene comprises a cargo nucleic acid sequence and a first and a second donor end sequences. As would be appreciated in the art, a donor often includes an open reading frame that encodes a transgene at the middle of donor and terminal repeat sequences at the 5’ and 3’ end of the donor. The translated helper (e.g., without limitation, the helper enzyme) binds to the 5’ and 3’ sequence of the donor and carries out the transposition function. In embodiments, a donor is used interchangeably with transposable elements, which are used to refer to polynucleotides capable of inserting copies of themselves into other polynucleotides. The term donor is well known to those skilled in the art and includes classes of donors that can be distinguished on the basis of sequence organization, for example inverted terminal sequences at each end, and / or directly repeated long terminal repeats (LTRs) at the ends. In embodiments, the donor as described herein may be described as a piggyBac like element, e.g., a donor element that is characterized by its traceless excision, which recognizes TTAA (SEQ ID NO: 440) 46 DB1 / 147031523.8 SAL-017PC / 126933-5017 sequence and restores the sequence at the insert site back to the original TTAA (SEQ ID NO: 440) sequence after removal of the donor. In embodiments, the donor is flanked by one or more end sequences or terminal ends. In embodiments, the donor is or comprises a gene encoding a complete polypeptide. In embodiments, the donor is or comprises a gene which is defective or substantially absent in a disease state. In embodiments, a transgene is associated with various regulatory elements that are selected to ensure stable expression of a construct with the transgene. Thus, in embodiments, a transgene is encoded by a non-viral vector (e.g., without limitation, a DNA plasmid) that can comprise one or more insulator sequences that prevent or mitigate activation or inactivation of nearby genes. The insulators flank the donor (transgene cassette) to reduce transcriptional silencing and position effects imparted by chromosomal sequences. As an additional effect, the insulators can eliminate functional interactions of the transgene enhancer and promoter sequences with neighboring chromosomal sequences. In embodiments, the one or more insulator sequences comprise an HS4 insulator (1.2-kb 5’-HS4 chicken β-globin (cHS4) insulator element) and an D4Z4 insulator (tandem macrosatellite repeats linked to Facio-Scapulo-Humeral Dystrophy (FSHD). In embodiments, the sequences of the HS4 insulator and the D4Z4 insulator are as described in Rival-Gervier et al. Mol Ther.2013 Aug; 21(8):1536-50, which is incorporated herein by reference in its entirety. In embodiments, the transgene is inserted into a GSHS location in a host genome. GSHSs is defined as loci well- suited for gene transfer, as integrations within these sites are not associated with adverse effects such as proto- oncogene activation, tumor suppressor inactivation, or insertional mutagenesis. GSHSs can defined by the following criteria: (1) distance of at least 50 kb from the 5’ end of any gene, (2) distance of at least 300 kb from any cancer-related gene, (3) distance of at least 300 kb from any microRNA (miRNA), (4) location outside a transcription unit, and (5) location outside ultra-conserved regions (UCRs) of the human genome. See Papapetrou et al. Nat Biotechnol 2011;29:73-8; Bejerano et al. Science 2004;304:1321-5. Furthermore, the use of GSHS locations can allow stable transgene expression across multiple cell types. One such site, chemokine C-C motif receptor 5 (CCR5) has been identified and used for integrative gene transfer. CCR5 is a member of the beta chemokine receptor family and is required for the entry of R5 tropic viral strains involved in primary infections. A homozygous 32 bp deletion in the CCR5 gene confers resistance to HIV-1 virus infections in humans. Disrupted CCR5 expression, naturally occurring in about 1% of the Caucasian population, does not appear to result in any reduction in immunity. Lobritz at al., Viruses 2010;2:1069-105. A clinical trial has demonstrated safety and efficacy of disrupting CCR5 via targetable nucleases. Tebas at al., HIV. N Engl J Med 2014;370:901-10. In embodiments, the CFTR transgene further comprises a promoter, optionally wherein the CFTR transgene targets a safe harbor site. In embodiments, the CFTR transgene does not comprise a promoter, optionally wherein the CFTR transgene targets the CFTR gene. 47 DB1 / 147031523.8 SAL-017PC / 126933-5017 Linkers In embodiments, the targeting element comprises a nucleic acid binding component of a gene-editing system. In embodiments, the enzyme or variant thereof and the targeting element are connected. In embodiments, the enzyme and the targeting element are fused to one another or linked via a linker to one another. In embodiments, the linker is a flexible linker. In embodiments, the flexible linker is substantially comprised of glycine and serine residues, optionally wherein the flexible linker comprises (Gly4Ser)n, where n is an integer from 1-12. In embodiments, the flexible linker is of about 20, or about 30, or about 40, or about 50, or about 60 amino acid residues. In embodiments, the flexible linker is about 50, or about 100, or about 150, or about 200 amino acid residues in length. In embodiments, the flexible linker comprises at least about 150 nucleotides (nt), or at least about 200 nt, or at least about 250 nt, or at least about 300 nt, or at least about 350 nt, or at least about 400 nt, or at least about 450 nt, or at least about 500 nt, or at least about 500 nt, or at least about 600 nt. In embodiments, the flexible linker comprises from about 450 nt to about 500 nt. In embodiments, the enzyme is directly fused to the N-terminus of the targeting element and, optionally, wherein the targeting element is or comprises a Zinc finger, TALE, and / or dCas9 enzyme. In embodiments, the enzyme or variant thereof is able to directly or indirectly cause transposition of a target gene. In embodiments, the enzyme or variant thereof is able to directly or indirectly interact and / or form a complex with one or more proteins or nucleic acids. In embodiments, the linker is or comprises (GSS)4or the linker is GS flanked on either side of a targeting element, optionally TALE and ZnF. In embodiments, the linker connects the targeting element of the N-terminus of the helper enzyme or connects the targeting element within the helper enzyme. In embodiment, the linker is a non-covalent linker. In embodiments, the non-covalent linker comprises an epitope tag. In embodiments, the non-covalent linker comprises an ALFA tag. In embodiments, the ALFA tag further comprises recombinant variable domains of heavy-chain-only antibodies (VHH) (NANOBODY). In embodiments, the non-covalent linker comprises recombinant variable domains of heavy-chain-only antibodies (VHH) (NANOBODY). In embodiments, the non-covalent linker comprises recombinant variable domains of heavy-chain- only antibodies (VHH) (NANOBODY) binding to ALFA tagged protein (NbALFA). In embodiments, the linker comprises a monobody. In embodiments, the linker comprises an ALFA tag and monobody. In embodiments, the ALFA tag comprises an amino acid sequence of SRLEEELRRRLTE (SEQ ID NO: 892), or a variant thereof having a substitution or deletion. In embodiments, the helper enzyme further comprises a linker inserted after a residue selected from S60-E70 relative to SEQ ID NO: 1. In embodiments, the helper enzyme further comprises a linker inserted after the G66 residue relative to SEQ ID NO: 1. In embodiments, the helper enzyme further comprises a linker inserted after the S60 residue relative to SEQ ID NO: 1. In embodiments, the helper enzyme further comprises a linker inserted after the E70 residue relative to SEQ ID NO: 1. In embodiments, the helper enzyme further comprises a linker inserted 48 DB1 / 147031523.8 SAL-017PC / 126933-5017 after the L80 residue relative to SEQ ID NO: 1. In embodiments, the helper enzyme further comprises a linker inserted after the N90 residue relative to SEQ ID NO: 1. In embodiments, the helper enzyme further comprises a linker inserted after the D100 residue relative to SEQ ID NO: 1. In embodiments, the linker is inserted immediately after residue G66 relative to SEQ ID NO: 1. Inteins Inteins (INTervening protEINS) are mobile genetic elements that are protein domains, found in nature, with the capability to carry out the process of protein splicing. See Sarmiento & Camarero (2019) Curr. Protein Pept. Sci., 20(5), 408–424, which is incorporated by reference herein in its entirety. Protein spicing is a post-translation biochemical modification which results in the cleavage and formation of peptide bonds between precursor polypeptide segments flanking the intein. Id. Inteins apply standard enzymatic strategies to excise themselves post-translationally from a precursor protein via protein splicing. Nanda et al., Microorganisms vol.8,122004.16 Dec.2020. An intein can splice its flanking N- and C-terminal domains to become a mature protein and excise itself from a sequence. For example, split inteins have been used to control the delivery of heterologous genes into transgenic organisms. See Wood & Camarero (2014) J Biol Chem.289(21):14512-14519. This approach relies on splitting the target protein into two segments, which are then post-translationally reconstituted in vivo by protein trans-splicing (PTS). See Aboye & Camarero (2012) J. Biol. Chem.287, 27026–27032. More recently, an intein- mediated split-Cas9 system has been developed to incorporate Cas9 into cells and reconstitute nuclease activity efficiently. Truong et al., Nucleic Acids Res.2015, 43 (13), 6450–6458. The protein splicing excises the internal region of the precursor protein, which is then followed by the ligation of the N-extein and C-extein fragments, resulting in two polypeptides – the excised intein and the new polypeptide produced by joining the C- and N-exteins. Sarmiento & Camarero (2019). In embodiments, intein-mediated incorporation of DNA binders such as, without limitation, dCas9, dCas12j, Zinc Fingers, or TALEs, allows creation of a split-enzyme system such as, without limitation, split transposase system, which permits reconstitution of the full-length enzyme, e.g., transposase, from two smaller fragments. This allows avoiding the need to express DNA binders at the N- or C-terminus of an enzyme, e.g., transposase. In this approach, the two portions of an enzyme, e.g., transposase, are fused to the intein and, after co-expression, the intein allows producing a full-length enzyme, e.g., transposase, by post-translation modification. Thus, in embodiments, a nucleic acid encoding the enzyme capable of targeted genomic integration by transposition comprises an intein. In embodiments, the nucleic acid encodes the enzyme in the form of first and second portions with the intein encoded between the first and second portions, such that the first and second portions are fused into a functional enzyme upon post-translational excision of the intein from the enzyme. In embodiments, an intein is a suitable ligand-dependent intein, for example, an intein selected from those described in U.S. Patent No.9,200,045; Mootz et al., J. Am. Chem. Soc.2002; 124, 9044-9045; Mootz et al., J. Am. Chem. Soc.2003; 125, 10561-10569; Buskirk et al., Proc. Natl. Acad. Sci. USA.2004; 101, 10505-10510; 49 DB1 / 147031523.8 SAL-017PC / 126933-5017 Skretas & Wood. Protein Sci.2005; 14, 523-532; Schwartz, et al., Nat. Chem. Biol.2007; 3, 50-54; Peck et al., Chem. Biol.2011; 18 (5), 619-630; the entire contents of each of which are hereby incorporated by reference herein. In embodiments, wherein a nucleic acid encoding the enzyme capable of targeted genomic integration by transposition comprises an intein, optionally NpuN (Intein-N) (SEQ ID NO: 423) and / or NpuC (Intein-C) (SEQ ID NO: 424), or a variant thereof. SEQ ID NO: 423: nucleotide sequence of NpuN (Intein-N) GGCGGATCTGGCGGTAGTGCTGAGTATTGTCTGAGTTACGAAACGGAAATACTCACGGTTGAGTATGGGCTTCTT CCAATTGGCAAAATCGTTGAAAAGCGCATAGAGTGTACGGTGTATTCCGTCGATAACAACGGTAATATCTACACC CAGCCGGTAGCTCAGTGGCACGACCGAGGCGAACAGGAAGTGTTCGAGTATTGCTTGGAAGATGGCTCCCTTATC CGCGCCACTAAAGACCATAAGTTTATGACGGTTGACGGGCAGATGCTGCCTATAGACGAAATATTTGAGAGAGAG CTGGACTTGATGAGAGTCGATAATCTGCCAAAT SEQ ID NO: 424: nucleotide sequence of NpuC (Intein-C) GGCGGATCTGGCGGTAGTGGGGGTTCCGGATCCATAAAGATAGCTACTAGGAAATATCTTGGCAAACAAAACGTC TATGACATAGGAGTTGAGCGAGATCACAATTTTGCTTTGAAGAATGGGTTCATCGCGTCTAATTGCTTCAACGCT AGCGGCGGGTCAGGAGGCTCTGGTGGAAGC In embodiments the intein is NpuN (Intein-N) (SEQ ID NO: 423) and / or NpuC (Intein-C) (SEQ ID NO: 424), or a variant thereof, e.g., a sequence having at least about 90%, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto. In embodiments, the nucleic acid encodes the enzyme in the form of first and second portions with the intein encoded between the first and second portions, such that the first and second portions are fused into a functional enzyme upon post-translational excision of the intein from the enzyme. In embodiments, the intein is suitable for linking the enzyme and the targeting element. In embodiments, a nucleic acid encoding the enzyme capable of targeted genomic integration by transposition comprises a dimerization enhancer. In embodiments, the nucleic acid encodes the enzyme in the form of first and second portions with the dimerization enhancer encoded between the first and second portions, such that the first and second portions are fused into a functional enzyme upon post-translational excision of the dimerization enhancer from the enzyme. In embodiments, the dimerization enhancer is suitable for linking the enzyme and the targeting element. In embodiments, the dimerization enhancer is selected from: a protein comprising a SH3 domain, biotin, avidin, or a rapamycin binder, optionally, wherein the rapamycin binder is FKBP12 or mTOR, or a variant thereof. In embodiments, the transgene comprises a nucleic acid sequence and a first and a second end sequences. In embodiments, the transgene is flanked by the first and the second end sequences. In embodiments, the end sequences are selected from nucleotide sequences of SEQ ID NO: 6 and / or SEQ ID NO: 7, or a nucleotide sequence having at least about 90% identity thereto. In embodiments, the end sequences include 50 DB1 / 147031523.8 SAL-017PC / 126933-5017 at least one repeat from a nucleotide sequence having at least about 90% identity to the nucleotide sequence of SEQ ID NO: 6. In embodiments, the at least one repeat from the nucleotide sequence having at least about 90% identity to the nucleotide sequence of SEQ ID NO: 6 is positioned at the 5’ end of the transgene. In embodiments, the end sequences further comprise at least one repeat from a nucleotide sequence having at least about 90% identity to the nucleotide sequence of SEQ ID NO: 7. In embodiments, the at least one repeat from the nucleotide sequence having at least about 90% identity to the nucleotide sequence of SEQ ID NO: 7 is positioned at the 3’ end of the transgene. In embodiments, the enzyme or variant thereof is incorporated into a vector or a vector-like particle. In embodiments, the vector or a vector-like particle comprises one or more expression cassettes. In embodiments, the vector or a vector-like particle comprises one expression cassette. In embodiments, the expression cassette further comprises the enzyme or variant thereof, the transgene, the end sequences, or a combination thereof. In embodiments, the enzyme or variant thereof, the transgene, the end sequences, or a combination thereof are incorporated into one or more vectors or vector-like particles. In embodiments, the enzyme or variant thereof, the transgene, the end sequences, or combination thereof are incorporated into a same vector or vector-like particle. In embodiments, the enzyme or variant thereof, the transgene, the end sequences, or combination thereof is incorporated into different vectors or vector-like particles. In embodiments, the vector or vector-like particle is nonviral. In embodiments, the transgene does not comprise a promoter, optionally wherein the transgene targets the CFTR gene. In embodiments, the transgene is under the control of at least one tissue-specific promoter. In embodiments, the transgene is under the control of at least one tissue-specific promoter, optionally wherein the transgene targets a safe harbor site. In embodiments, the tissue-specific promoter is a ciliated cell-specific promoter. In embodiments, the ciliated cell-specific promoter targets the expression of a transgene to the airway surface epithelium. In embodiments, the at least one tissue-specific promoter is a single promoter. In embodiments, the at least one tissue-specific promoter is under the control of a dual promoter or a tandem promoter. In embodiments, the transgene comprises peptides for linking fragments thereof. In embodiments, the transgene is linked to polynucleotide comprising a sequence comprising a 5’-miRNA, a sense and antisense miRNA pair, and / or a 3’-miRNA. In embodiments, the composition comprises DNA, RNA, or both. In embodiments, the enzyme or variant thereof is in the form of RNA. Host Cell In aspects, the present disclosure further provides a host cell comprising the composition in accordance with embodiments of the present disclosure. 51 DB1 / 147031523.8 SAL-017PC / 126933-5017 Lipid Nanoparticle (LNP) In embodiments, the present disclosure provides a composition, wherein the composition is encapsulated in a lipid nanoparticle (LNP). In embodiments, the polynucleotide encoding the enzyme or variant thereof and the polynucleotide encoding the transgene are in the form of the same LNP, optionally in a co-formulation. In embodiments, the LNP comprises one or more lipids selected from 1,2-dioleoyl-3-trimethylammonium propane (DOTAP), a cationic cholesterol derivative mixed with dimethylaminoethane-carbamoyl (DC-Chol), phosphatidylcholine (PC), triolein (glyceryl trioleate), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [carboxy(polyethylene glycol)-2000] (DSPE-PEG), 1,2-dimyristoyl-rac-glycero-3-methoxypolyethyleneglycol – 2000 (DMG-PEG 2K), and 1,2 distearol -sn-glycerol-3phosphocholine (DSPC) and / or comprising of one or more molecules selected from polyethylenimine (PEI) and poly(lactic-co-glycolic acid) (PLGA), N-Acetylgalactosamine (GalNAc), and SSOP LNPs. In embodiments, the LNP targets the lung. Methods of Treatment In embodiments, the present disclosure provides a method for treating and / or mitigating cystic fibrosis (CF), comprising administering to a patient in need thereof a composition of the present disclosure. In embodiments, the present disclosure provides a method for treating and / or mitigating cystic fibrosis (CF), comprising: (a) contacting a cell obtained from a patient with a composition of the present disclosure; and (b) administering the cell to a patient in need thereof. In embodiments, the CF is characterized by one or more mutations in a CFTR gene. In embodiments, the CFTR mutation comprises deficiency in protein production, protein processing, gating, conduction, and / or insufficient protein. In embodiments, the CFTR mutation is selected from: G542X, W1282X, R553X, F508del, N1303K, I507del, G551D, S549N, D1152H, R347P, R117H, 3849+10kbC→T, 2789+5G→A, and A455E. CF Treatments In embodiments, the method of the present disclosure improves or corrects defective membrane trafficking relative to an untreated patient or the patient at the start of treatment. In embodiments, the method of the present disclosure improves or reestablishes normal chloride transport relative to an untreated patient or the patient at the start of treatment. In embodiments, the method of the present disclosure reduces or inhibits mucus hypersecretion relative to an untreated patient or the patient at the start of treatment. 52 DB1 / 147031523.8 SAL-017PC / 126933-5017 In embodiments, the method of the present disclosure improves or reestablishes mucus hydration in airways relative to an untreated patient or the patient at the start of treatment. In embodiments, the method of the present disclosure improves or reestablishes airway surface liquid pH relative to an untreated patient or the patient at the start of treatment. In embodiments, the method of the present disclosure reduces or inhibits airway infection relative to an untreated patient or the patient at the start of treatment. In embodiments, the method of the present disclosure reduces or inhibits airway obstruction relative to an untreated patient or the patient at the start of treatment. In embodiments, the method of the present disclosure improves or reestablishes pancreatic insulin production relative to an untreated patient or the patient at the start of treatment. In embodiments, the method of the present disclosure improves or reestablishes intestinal motility relative to an untreated patient or the patient at the start of treatment. In embodiments, the method of the present disclosure improves or reestablishes intestinal absorption of nutrients relative to an untreated patient or the patient at the start of treatment. In embodiments, the method of the present disclosure is substantially non-immunogenic. In embodiments, the method of the present disclosure provides a method wherein the treatment and / or mitigation is durable. In embodiments, the method of the present disclosure requires a single administration. In embodiments, the method of the present disclosure provides a method wherein the administering is intravenous. In embodiments, the method of the present disclosure provides a method wherein the administering is by inhalation. In embodiments, the method of the present disclosure comprises administering to the patient in need thereof an additional therapeutic agent. In embodiments, the additional therapeutic agent is optionally selected from one or more of a KALYDECO (ivacaftor), ORKAMBI (lumacaftor / ivacaftor), SYMDEKO (tezacaftor / ivacaftor), and TRIKAFTA (elexacaftor / tezacaftor / ivacaftor). In embodiments, there is provided a host cell comprising a composition as described herein (e.g., without limitation, a composition comprising the gene transfer construct and / or transposase). In embodiments, the host cell is a prokaryotic or eukaryotic cell, e.g., a mammalian cell. In embodiments, there is provided a transgenic organism that may comprise cells which have been transformed by the methods of the present disclosure. In one example, the organism may be a mammal or an insect. When the organism is a mammal, the organism may include, but is not limited to, a mouse, a cat, a monkey, a dog, a brown bear, a panda, a chimpanzee, and the like. When the organism is an insect, the organism may include, but is not limited to, a fruit fly, a mosquito, a butterfly, a bollworm and the like. 53 DB1 / 147031523.8 SAL-017PC / 126933-5017 The compositions can be included in a container, kit, pack, or dispenser together with instructions for administration. Also provided herein are kits comprising: i) any of the aforementioned gene transfer constructs of this disclosure, and / or any of the aforementioned cells of this disclosure and ii) a container. In certain embodiments, the kits further comprise instructions for the use thereof. In certain embodiments, any of the aforementioned kits can further comprise a recombinant DNA construct comprising a nucleic acid sequence that encodes a transposase. DEFINITIONS The following definitions are used in connection with the disclosure disclosed herein. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of skill in the art to which this disclosure belongs. The term “in vivo” refers to an event that takes place in a subject’s body. The term “ex vivo” refers to an event which involves treating or performing a procedure on a cell, tissue and / or organ which has been removed from a subject’s body. Aptly, the cell, tissue and / or organ may be returned to the subject’s body in a method of treatment or surgery. As used herein, the term “variant” encompasses but is not limited to nucleic acids or proteins which comprise a nucleic acid or amino acid sequence which differs from the nucleic acid or amino acid sequence of a reference by way of one or more substitutions, deletions and / or additions at certain positions. The variant may comprise one or more conservative substitutions. Conservative substitutions may involve, e.g., the substitution of similarly charged or uncharged amino acids. “Carrier” or “vehicle” as used herein refer to carrier materials suitable for drug administration. Carriers and vehicles useful herein include any such materials known in the art, e.g., any liquid, gel, solvent, liquid diluent, solubilizer, surfactant, or the like, which is nontoxic and which does not interact with other components of the composition in a deleterious manner. The phrase “pharmaceutically acceptable” refers to those compounds, materials, compositions, and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problems or complications commensurate with a reasonable benefit / risk ratio. The terms “pharmaceutically acceptable carrier” or “pharmaceutically acceptable excipient” are intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and inert ingredients. The use of such pharmaceutically acceptable carriers or pharmaceutically acceptable excipients for active pharmaceutical ingredients is well known in the art. Except insofar as any conventional pharmaceutically acceptable carrier or pharmaceutically acceptable excipient is incompatible with the active pharmaceutical ingredient, its use in the therapeutic compositions of the disclosure is contemplated. 54 DB1 / 147031523.8 SAL-017PC / 126933-5017 Additional active pharmaceutical ingredients, such as other drugs, can also be incorporated into the described compositions and methods. As used herein, “a,” “an,” or “the” can mean one or more than one. Further, the term “about” when used in connection with a referenced numeric indication means the referenced numeric indication plus or minus up to 10% of that referenced numeric indication. For example, the language “about 50” covers the range of 45 to 55. As used herein, the word “include,” and its variants, is intended to be non-limiting, such that recitation of items in a list is not to the exclusion of other like items that may also be useful in the compositions and methods of this technology. Similarly, the terms “can” and “may” and their variants are intended to be non-limiting, such that recitation that an embodiment can or may comprise certain elements or features does not exclude other embodiments of the present technology that do not contain those elements or features. Although the open-ended term “comprising,” as a synonym of terms such as including, containing, or having, is used herein to describe and claim the invention, the present invention, or embodiments thereof, may alternatively be described using alternative terms such as “consisting of” or “consisting essentially of.” As used herein, the words “preferred” and “preferably” refer to embodiments of the technology that afford certain benefits, under certain circumstances. However, other embodiments may also be preferred, under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful, and is not intended to exclude other embodiments from the scope of the technology. SELECTED SEQUENCES In embodiments, the present disclosure provides for any of the sequence provided herein, including the below, and a variant sequence having at least about 90%, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto, or at least about 10 mutations, or at least about 9 mutations, or at least about 8 mutations, or at least about 7 mutations, or at least about 6 mutations, or at least about 5 mutations, or at least about 4 mutations, or at least about 3 mutations, or at least about 2 mutations, or at least about 1 mutation. SEQ ID NO: 1: amino acid sequence of a variant of the hyperactive helper with S at position 8 and C at position 13 (572 amino acids) 1 MAQHSDYSDD EFCADKLSNY SCDSDLENAS TSDEDSSDDE VMVRPRTLRR RRISSSSSDS 61 ESDIEGGREE WSHVDNPPVL EDFLGHQGLN TDAVINNIED AVKLFIGDDF FEFLVEESNR 121 YYNQNRNNFK LSKKSLKWKD ITPQEMKKFL GLIVLMGQVR KDRRDDYWTT EPWTETPYFG 181 KTMTRDRFRQ IWKAWHFNNN ADIVNESDRL CKVRPVLDYF VPKFINIYKP HQQLSLDEGI 241 VPWRGRLFFR VYNAGKIVKY GILVRLLCES DTGYICNMEI YCGEGKRLLE TIQTVVSPYT 301 DSWYHIYMDN YYNSVANCEA LMKNKFRICG TIRKNRGIPK DFQTISLKKG ETKFIRKNDI 361 LLQVWQSKKP VYLISSIHSA EMEESQNIDR TSKKKIVKPN ALIDYNKHMK GVDRADQYLS 421 YYSILRRTVK WTKRLAMYMI NCALFNSYAV YKSVRQRKMG FKMFLKQTAI HWLTDDIPED 481 MDIVPDLQPV PSTSGMRAKP PTSDPPCRLS MDMRKHTLQA IVGSGKKKNI LRRCRVCSVH 541 KLRSETRYMC KFCNIPLHKG ACFEKYHTLK NY 55 DB1 / 147031523.8 SAL-017PC / 126933-5017 SEQ ID NO: 2: nucleotide sequence encoding SEQ ID NO: 1 (1719 nt) 1 ATGGCCCAGC ACAGCGACTA CCCCGACGAC GAGTTCAGAG CCGATAAGCT GAGTAACTAC 61 AGCTGCGACA GCGACCTGGA AAACGCCAGC ACATCCGACG AGGACAGCTC TGACGACGAG 121 GTGATGGTGC GGCCCAGAAC CCTGAGACGG AGAAGAATCA GCAGCTCTAG CAGCGACTCT 181 GAATCCGACA TCGAGGGCGG CCGGGAAGAG TGGAGCCACG TGGACAACCC TCCTGTTCTG 241 GAAGATTTTC TGGGCCATCA GGGCCTGAAC ACCGACGCCG TGATCAACAA CATCGAGGAT 301 GCCGTGAAGC TGTTCATAGG AGATGATTTC TTTGAGTTCC TGGTCGAGGA ATCCAACCGC 361 TATTACAACC AGAATAGAAA CAACTTCAAG CTGAGCAAGA AAAGCCTGAA GTGGAAGGAC 421 ATCACCCCTC AGGAGATGAA AAAGTTCCTG GGACTGATCG TTCTGATGGG ACAGGTGCGG 481 AAGGACAGAA GGGATGATTA CTGGACAACC GAACCTTGGA CCGAGACCCC TTACTTTGGC 541 AAGACCATGA CCAGAGACAG ATTCAGACAG ATCTGGAAAG CCTGGCACTT CAACAACAAT 601 GCTGATATCG TGAACGAGTC TGATAGACTG TGTAAAGTGC GGCCAGTGTT GGATTACTTC 661 GTGCCTAAGT TCATCAACAT CTATAAGCCT CACCAGCAGC TGAGCCTGGA TGAAGGCATC 721 GTGCCCTGGC GGGGCAGACT GTTCTTCAGA GTGTACAATG CTGGCAAGAT CGTCAAATAC 781 GGCATCCTGG TGCGCCTTCT GTGCGAGAGC GATACAGGCT ACATCTGTAA TATGGAAATC 841 TACTGCGGCG AGGGCAAAAG ACTGCTGGAA ACCATCCAGA CCGTCGTTTC CCCTTATACC 901 GACAGCTGGT ACCACATCTA CATGGACAAC TACTACAATT CTGTGGCCAA CTGCGAGGCC 961 CTGATGAAGA ACAAGTTTAG AATCTGCGGC ACAATCAGAA AAAACAGAGG CATCCCTAAG 1021 GACTTCCAGA CCATCTCTCT GAAGAAGGGC GAAACCAAGT TCATCAGAAA GAACGACATC 1081 CTGCTCCAAG TGTGGCAGTC CAAGAAACCC GTGTACCTGA TCAGCAGCAT CCATAGCGCC 1141 GAGATGGAAG AAAGCCAGAA CATCGACAGA ACAAGCAAGA AGAAGATCGT GAAGCCCAAT 1201 GCTCTGATCG ACTACAACAA GCACATGAAA GGCGTGGACC GGGCCGACCA GTACCTGTCT 1261 TATTACTCTA TCCTGAGAAG AACAGTGAAA TGGACCAAGA GACTGGCCAT GTACATGATC 1321 AATTGCGCCC TGTTCAACAG CTACGCCGTG TACAAGTCCG TGCGACAAAG AAAAATGGGA 1381 TTCAAGATGT TCCTGAAGCA GACAGCCATC CACTGGCTGA CAGACGACAT TCCTGAGGAC 1441 ATGGACATTG TGCCAGATCT GCAACCTGTG CCCAGCACCT CTGGTATGAG AGCTAAGCCT 1501 CCCACCAGCG ATCCTCCATG TAGACTGAGC ATGGACATGC GGAAGCACAC CCTGCAGGCC 1561 ATCGTCGGCA GCGGCAAGAA GAAGAACATC CTTAGACGGT GCAGGGTGTG CAGCGTGCAC 1621 AAGCTGCGGA GCGAGACTCG GTACATGTGC AAGTTTTGCA ACATTCCCCT GCACAAGGGA 1681 GCCTGCTTCG AGAAGTACCA CACCCTGAAG AATTACTAG SEQ ID NO: 3: nucleotide sequence of hyperactive helper mRNA helper construct (1956 bp) (Order of underlined sequences: T7 promoter, hyperactive helper, polyA tail; the 5’-globin and 3’-globin UTRs are in capital letters). 1 taatacgact cactataagg aagCTTCTTG TTCTTTTTGC AGAAGCTCAG AATAAACGCT 61 CAACTTTGGc cgccaccatg gcccagcaca gcgactaccc cgacgacgag ttcagagccg 121 ataagctgag taactacagc tgcgacagcg acctggaaaa cgccagcaca tccgacgagg 181 acagctctga cgacgaggtg atggtgcggc ccagaaccct gagacggaga agaatcagca 241 gctctagcag cgactctgaa tccgacatcg agggcggccg ggaagagtgg agccacgtgg 301 acaaccctcc tgttctggaa gattttctgg gccatcaggg cctgaacacc gacgccgtga 361 tcaacaacat cgaggatgcc gtgaagctgt tcataggaga tgatttcttt gagttcctgg 421 tcgaggaatc caaccgctat tacaaccaga atagaaacaa cttcaagctg agcaagaaaa 481 gcctgaagtg gaaggacatc acccctcagg agatgaaaaa gttcctggga ctgatcgttc 541 tgatgggaca ggtgcggaag gacagaaggg atgattactg gacaaccgaa ccttggaccg 601 agacccctta ctttggcaag accatgacca gagacagatt cagacagatc tggaaagcct 661 ggcacttcaa caacaatgct gatatcgtga acgagtctga tagactgtgt aaagtgcggc 721 cagtgttgga ttacttcgtg cctaagttca tcaacatcta taagcctcac cagcagctga 781 gcctggatga aggcatcgtg ccctggcggg gcagactgtt cttcagagtg tacaatgctg 841 gcaagatcgt caaatacggc atcctggtgc gccttctgtg cgagagcgat acaggctaca 901 tctgtaatat ggaaatctac tgcggcgagg gcaaaagact gctggaaacc atccagaccg 961 tcgtttcccc ttataccgac agctggtacc acatctacat ggacaactac tacaattctg 1021 tggccaactg cgaggccctg atgaagaaca agtttagaat ctgcggcaca atcagaaaaa 1081 acagaggcat ccctaaggac ttccagacca tctctctgaa gaagggcgaa accaagttca 1141 tcagaaagaa cgacatcctg ctccaagtgt ggcagtccaa gaaacccgtg tacctgatca 1201 gcagcatcca tagcgccgag atggaagaaa gccagaacat cgacagaaca agcaagaaga 1261 agatcgtgaa gcccaatgct ctgatcgact acaacaagca catgaaaggc gtggaccggg 1321 ccgaccagta cctgtcttat tactctatcc tgagaagaac agtgaaatgg accaagagac 1381 tggccatgta catgatcaat tgcgccctgt tcaacagcta cgccgtgtac aagtccgtgc 56 DB1 / 147031523.8 SAL-017PC / 126933-5017 1441 gacaaagaaa aatgggattc aagatgttcc tgaagcagac agccatccac tggctgacag 1501 acgacattcc tgaggacatg gacattgtgc cagatctgca acctgtgccc agcacctctg 1561 gtatgagagc taagcctccc accagcgatc ctccatgtag actgagcatg gacatgcgga 1621 agcacaccct gcaggccatc gtcggcagcg gcaagaagaa gaacatcctt agacggtgca 1681 gggtgtgcag cgtgcacaag ctgcggagcg agactcggta catgtgcaag ttttgcaaca 1741 ttcccctgca caagggagcc tgcttcgaga agtaccacac cctgaagaat tactagAACC 1801 AGCCTCAAGA ACACCCGAAT GGAGTCTCTA AGCTACATAA TACCAACTTA CACTTTACAA 1861 AATGTTGTCC CCCAAAATGT AGCCATTCGT ATCTGCTCCT AATAAAAAGA AAGTTTCTTC 1921 ACaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaa SEQ ID NO: 4: amino acid sequence of hyperactive helper (572 amino acids) 1 MAQHSDYPDD EFRADKLSNY SCDSDLENAS TSDEDSSDDE VMVRPRTLRR RRISSSSSDS 61 ESDIEGGREE WSHVDNPPVL EDFLGHQGLN TDAVINNIED AVKLFIGDDF FEFLVEESNR 121 YYNQNRNNFK LSKKSLKWKD ITPQEMKKFL GLIVLMGQVR KDRRDDYWTT EPWTETPYFG 181 KTMTRDRFRQ IWKAWHFNNN ADIVNESDRL CKVRPVLDYF VPKFINIYKP HQQLSLDEGI 241 VPWRGRLFFR VYNAGKIVKY GILVRLLCES DTGYICNMEI YCGEGKRLLE TIQTVVSPYT 301 DSWYHIYMDN YYNSVANCEA LMKNKFRICG TIRKNRGIPK DFQTISLKKG ETKFIRKNDI 361 LLQVWQSKKP VYLISSIHSA EMEESQNIDR TSKKKIVKPN ALIDYNKHMK GVDRADQYLS 421 YYSILRRTVK WTKRLAMYMI NCALFNSYAV YKSVRQRKMG FKMFLKQTAI HWLTDDIPED 481 MDIVPDLQPV PSTSGMRAKP PTSDPPCRLS MDMRKHTLQA IVGSGKKKNI LRRCRVCSVH 541 KLRSETRYMC KFCNIPLHKG ACFEKYHTLK NY SEQ ID NO: 5: nucleotide sequence encoding hyperactive helper (SEQ ID NO: 4) (1719 nt) 1 ATGGCCCAGC ACAGCGACTA CCCCGACGAC GAGTTCAGAG CCGATAAGCT GAGTAACTAC SAL-017PC / 126933-5017 61 RRISSSSSDS ESDIEGGREE WSHVDNPPVL EDFLGHQGLN TDAVINNIED AVKLFIGDDF 121 FEFLVEESNR YYNQNRNNFK LSKKSLKWKD ITPQEMKKFL GLIVLMGQVR KDRRDDYWTT 181 EPWTETPYFG KTMTRDRFRQ IWKAWHFNNN ADIVNESDRL CKVRPVLDYF VPKFINIYKP 241 HQQLSLDEGI VPWRGRLFFR VYNAGKIVKY GILVRLLCES DTGYICNMEI YCGEGKRLLE 301 TIQTVVSPYT DSWYHIYMDN YYNSVANCEA LMKNKFRICG TIRKNRGIPK DFQTISLKKG 361 ETKFIRKNDI LLQVWQSKKP VYLISSIHSA EMEESQNIDR TSKKKIVKPN ALIDYNKHMK 421 GVDRADQYLS YYSILRRTVK WTKRLAMYMI NCALFNSYAV YKSVRQRKMG FKMFLKQTAI 481 HWLTDDIPED MDIVPDLQPV PSTSGMRAKP PTSDPPCRLS MDMRKHTLQA IVGSGKKKNI 541 LRRCRVCSVH KLRSETRYMC KFCNIPLHKG ACFEKYHTLK NY SEQ ID NO 16. Nucleotide sequence (1746 bp) of Saliogase “wild type” helper with NLS that when combined with shorted left ends (LE65, LE88, or LE90). Results in increase enzyme activity. 1 atggcgccta agaagaagag aaaggtcggc ggcgcccagc acagcgacta ctccgacgac 61 gagttctgcg ccgataagct gagtaactac agctgcgaca gcgacctgga aaacgccagc 121 acatccgacg aggacagctc tgacgacgag gtgatggtgc ggcccagaac cctgagacgg 181 agaagaatca gcagctctag cagcgactct gaatccgaca tcgagggcgg ccgggaagag 241 tggagccacg tggacaaccc tcctgttctg gaagattttc tgggccatca gggcctgaac 301 accgacgccg tgatcaacaa catcgaggat gccgtgaagc tgttcatagg agatgatttc 361 tttgagttcc tggtcgagga atccaaccgc tattacaacc agaatagaaa caacttcaag 421 ctgagcaaga aaagcctgaa gtggaaggac atcacccctc aggagatgaa aaagttcctg 481 ggactgatcg ttctgatggg acaggtgcgg aaggacagaa gggatgatta ctggacaacc 541 gaaccttgga ccgagacccc ttactttggc aagaccatga ccagagacag attcagacag 601 atctggaaag cctggcactt caacaacaat gctgatatcg tgaacgagtc tgatagactg 661 tgtaaagtgc ggccagtgtt ggattacttc gtgcctaagt tcatcaacat ctataagcct 721 caccagcagc tgagcctgga tgaaggcatc gtgccctggc ggggcagact gttcttcaga 781 gtgtacaatg ctggcaagat cgtcaaatac ggcatcctgg tgcgccttct gtgcgagagc 841 gatacaggct acatctgtaa tatggaaatc tactgcggcg agggcaaaag actgctggaa 901 accatccaga ccgtcgtttc cccttatacc gacagctggt accacatcta catggacaac 961 tactacaatt ctgtggccaa ctgcgaggcc ctgatgaaga acaagtttag aatctgcggc 1021 acaatcagaa aaaacagagg catccctaag gacttccaga ccatctctct gaagaagggc 1081 gaaaccaagt tcatcagaaa gaacgacatc ctgctccaag tgtggcagtc caagaaaccc 1141 gtgtacctga tcagcagcat ccatagcgcc gagatggaag aaagccagaa catcgacaga 1201 acaagcaaga agaagatcgt gaagcccaat gctctgatcg actacaacaa gcacatgaaa 1261 ggcgtggacc gggccgacca gtacctgtct tattactcta tcctgagaag aacagtgaaa 1321 tggaccaaga gactggccat gtacatgatc aattgcgccc tgttcaacag ctacgccgtg 1381 tacaagtccg tgcgacaaag aaaaatggga ttcaagatgt tcctgaagca gacagccatc 1441 cactggctga cagacgacat tcctgaggac atggacattg tgccagatct gcaacctgtg 1501 cccagcacct ctggtatgag agctaagcct cccaccagcg atcctccatg tagactgagc 1561 atggacatgc ggaagcacac cctgcaggcc atcgtcggca gcggcaagaa gaagaacatc 1621 cttagacggt gcagggtgtg cagcgtgcac aagctgcgga gcgagactcg gtacatgtgc 1681 aagttttgca acattcccct gcacaaggga gcctgcttcg agaagtacca caccctgaag 1741 aattac SEQ ID NO: 6: hyperactive helper Left ITR (157 bp) The left ITR retains recognition activity when the underlined nucleotides are deleted (80 bp). 1 ttaacacttg gattgcggga aacgagttaa gtcggctcgc gtgaattgcg cgtactccgc 61 gggagccgtc ttaactcggt tcatatagat ttgcggtgga gtgcgggaaa cgtgtaaact 121 cgggccgatt gtaactgcgt attaccaaat atttgtt SEQ ID NO: 7: hyperactive helper Right ITR (212 bp) The right ITR retains recognition activity when the underlined nucleotides are deleted (80 bp). 1 aattatttat gtactgaata gataaaaaaa tgtctgtgat tgaataaatt ttcatttttt 61 acacaagaaa ccgaaaattt catttcaatc gaacccatac ttcaaaagat ataggcattt 121 taaactaact ctgattttgc gcgggaaacc taaataattg cccgcgccat cttatatttt 58 DB1 / 147031523.8 SAL-017PC / 126933-5017 181 ggcgggaaat tcacccgaca ccgtagtgtt aa SEQ ID NO: 8: nucleotide sequence of dead Cas9 DNA binding protein (5004 bp) 1 ATGGACAAGA AGTACTCCAT TGGGCTCGCT ATCGGCACAA ACAGCGTCGG CTGGGCCGTC 61 ATTACGGACG AGTACAAGGT GCCGAGCAAA AAATTCAAAG TTCTGGGCAA TACCGATCGC 121 CACAGCATAA AGAAGAACCT CATTGGCGCC CTCCTGTTCG ACTCCGGGGA GACGGCCGAA 181 GCCACGCGGC TCAAAAGAAC AGCACGGCGC AGATATACCC GCAGAAAGAA TCGGATCTGC 241 TACCTGCAGG AGATCTTTAG TAATGAGATG GCTAAGGTGG ATGACTCTTT CTTCCATAGG 301 CTGGAGGAGT CCTTTTTGGT GGAGGAGGAT AAAAAGCACG AGCGCCACCC AATCTTTGGC 361 AATATCGTGG ACGAGGTGGC GTACCATGAA AAGTACCCAA CCATATATCA TCTGAGGAAG 421 AAGCTTGTAG ACAGTACTGA TAAGGCTGAC TTGCGGTTGA TCTATCTCGC GCTGGCGCAT 481 ATGATCAAAT TTCGGGGACA CTTCCTCATC GAGGGGGACC TGAACCCAGA CAACAGCGAT 541 GTCGACAAAC TCTTTATCCA ACTGGTTCAG ACTTACAATC AGCTTTTCGA AGAGAACCCG 601 ATCAACGCAT CCGGAGTTGA CGCCAAAGCA ATCCTGAGCG CTAGGCTGTC CAAATCCCGG 661 CGGCTCGAAA ACCTCATCGC ACAGCTCCCT GGGGAGAAGA AGAACGGCCT GTTTGGTAAT 721 CTTATCGCCC TGTCACTCGG GCTGACCCCC AACTTTAAAT CTAACTTCGA CCTGGCCGAA 781 GATGCCAAGC TTCAACTGAG CAAAGACACC TACGATGATG ATCTCGACAA TCTGCTGGCC 841 CAGATCGGCG ACCAGTACGC AGACCTTTTT TTGGCGGCAA AGAACCTGTC AGACGCCATT 901 CTGCTGAGTG ATATTCTGCG AGTGAACACG GAGATCACCA AAGCTCCGCT GAGCGCTAGT 961 ATGATCAAGC GCTATGATGA GCACCACCAA GACTTGACTT TGCTGAAGGC CCTTGTCAGA 1021 CAGCAACTGC CTGAGAAGTA CAAGGAAATT TTCTTCGATC AGTCTAAAAA TGGCTACGCC 1081 GGATACATTG ACGGCGGAGC AAGCCAGGAG GAATTTTACA AATTTATTAA GCCCATCTTG 1141 GAAAAAATGG ACGGCACCGA GGAGCTGCTG GTAAAGCTTA ACAGAGAAGA TCTGTTGCGC 1201 AAACAGCGCA CTTTCGACAA TGGAAGCATC CCCCACCAGA TTCACCTGGG CGAACTGCAC 1261 GCTATCCTCA GGCGGCAAGA GGATTTCTAC CCCTTTTTGA AAGATAACAG GGAAAAGATT 1321 GAGAAAATCC TCACATTTCG GATACCCTAC TATGTAGGCC CCCTCGCCCG GGGAAATTCC 1381 AGATTCGCGT GGATGACTCG CAAATCAGAA GAGACCATCA CTCCCTGGAA CTTCGAGGAA 1441 GTCGTGGATA AGGGGGCCTC TGCCCAGTCC TTCATCGAAA GGATGACTAA CTTTGATAAA 1501 AATCTGCCTA ACGAAAAGGT GCTTCCTAAA CACTCTCTGC TGTACGAGTA CTTCACAGTT 1561 TATAACGAGC TCACCAAGGT CAAATACGTC ACAGAAGGGA TGAGAAAGCC AGCATTCCTG 1621 TCTGGAGAGC AGAAGAAAGC TATCGTGGAC CTCCTCTTCA AGACGAACCG GAAAGTTACC 1681 GTGAAACAGC TCAAAGAAGA CTATTTCAAA AAGATTGAAT GTTTCGACTC TGTTGAAATC 1741 AGCGGAGTGG AGGATCGCTT CAACGCATCC CTGGGAACGT ATCACGATCT CCTGAAAATC 1801 ATTAAAGACA AGGACTTCCT GGACAATGAG GAGAACGAGG ACATTCTTGA GGACATTGTC 1861 CTCACCCTTA CGTTGTTTGA AGATAGGGAG ATGATTGAAG AACGCTTGAA AACTTACGCT 1921 CATCTCTTCG ACGACAAAGT CATGAAACAG CTCAAGAGGC GCCGATATAC AGGATGGGGG 1981 CGGCTGTCAA GAAAACTGAT CAATGGGATC CGAGACAAGC AGAGTGGAAA GACAATCCTG 2041 GATTTTCTTA AGTCCGATGG ATTTGCCAAC CGGAACTTCA TGCAGTTGAT CCATGATGAC 2101 TCTCTCACCT TTAAGGAGGA CATCCAGAAA GCACAAGTTT CTGGCCAGGG GGACAGTCTT 2161 CACGAGCACA TCGCTAATCT TGCAGGTAGC CCAGCTATCA AAAAGGGAAT ACTGCAGACC 2221 GTTAAGGTCG TGGATGAACT CGTCAAAGTA ATGGGAAGGC ATAAGCCCGA GAATATCGTT 2281 ATCGAGATGG CCCGAGAGAA CCAAACTACC CAGAAGGGAC AGAAGAACAG TAGGGAAAGG 2341 ATGAAGAGGA TTGAAGAGGG TATAAAAGAA CTGGGGTCCC AAATCCTTAA GGAACACCCA 2401 GTTGAAAACA CCCAGCTTCA GAATGAGAAG CTCTACCTGT ACTACCTGCA GAACGGCAGG 2461 GACATGTACG TGGATCAGGA ACTGGACATC AATCGGCTCT CCGACTACGA CGTGGCTGCT 2521 ATCGTGCCCC AGTCTTTTCT CAAAGATGAT TCTATTGATA ATAAAGTGTT GACAAGATCC 2581 GATAAAGCTA GAGGGAAGAG TGATAACGTC CCCTCAGAAG AAGTTGTCAA GAAAATGAAA 2641 AATTATTGGC GGCAGCTGCT GAACGCCAAA CTGATCACAC AACGGAAGTT CGATAATCTG 2701 ACTAAGGCTG AACGAGGTGG CCTGTCTGAG TTGGATAAAG CCGGCTTCAT CAAAAGGCAG 2761 CTTGTTGAGA CACGCCAGAT CACCAAGCAC GTGGCCCAAA TTCTCGATTC ACGCATGAAC 2821 ACCAAGTACG ATGAAAATGA CAAACTGATT CGAGAGGTGA AAGTTATTAC TCTGAAGTCT 2881 AAGCTGGTCT CAGATTTCAG AAAGGACTTT CAGTTTTATA AGGTGAGAGA GATCAACAAT 2941 TACCACCATG CGCATGATGC CTACCTGAAT GCAGTGGTAG GCACTGCACT TATCAAAAAA 3001 TATCCCAAGC TTGAATCTGA ATTTGTTTAC GGAGACTATA AAGTGTACGA TGTTAGGAAA 3061 ATGATCGCAA AGTCTGAGCA GGAAATAGGC AAGGCCACCG CTAAGTACTT CTTTTACAGC 3121 AATATTATGA ATTTTTTCAA GACCGAGATT ACACTGGCCA ATGGAGAGAT TCGGAAGCGA 3181 CCACTTATCG AAACAAACGG AGAAACAGGA GAAATCGTGT GGGACAAGGG TAGGGATTTC 3241 GCGACAGTCC GGAAGGTCCT GTCCATGCCG CAGGTGAACA TCGTTAAAAA GACCGAAGTA 3301 CAGACCGGAG GCTTCTCCAA GGAAAGTATC CTCCCGAAAA GGAACAGCGA CAAGCTGATC 59 DB1 / 147031523.8 SAL-017PC / 126933-5017 3361 GCACGCAAAA AAGATTGGGA CCCCAAGAAA TACGGCGGAT TCGATTCTCC TACAGTCGCT 3421 TACAGTGTAC TGGTTGTGGC CAAAGTGGAG AAAGGGAAGT CTAAAAAACT CAAAAGCGTC 3481 AAGGAACTGC TGGGCATCAC AATCATGGAG CGATCAAGCT TCGAAAAAAA CCCCATCGAC 3541 TTTCTGGAGG CGAAAGGATA TAAAGAGGTC AAAAAAGACC TCATCATTAA GCTTCCCAAG 3601 TACTCTCTCT TTGAGCTTGA AAACGGCCGG AAACGAATGC TCGCTAGTGC GGGCGAGCTG 3661 CAGAAAGGTA ACGAGCTGGC ACTGCCCTCT AAATACGTTA ATTTCTTGTA TCTGGCCAGC 3721 CACTATGAAA AGCTCAAAGG GTCTCCCGAA GATAATGAGC AGAAGCAGCT GTTCGTGGAA 3781 CAACACAAAC ACTACCTTGA TGAGATCATC GAGCAAATAA GCGAATTCTC CAAAAGAGTG 3841 ATCCTCGCCG ACGCTAACCT CGATAAGGTG CTTTCTGCTT ACAATAAGCA CAGGGATAAG 3901 CCCATCAGGG AGCAGGCAGA AAACATTATC CACTTGTTTA CTCTGACCAA CTTGGGCGCG 3961 CCTGCAGCCT TCAAGTACTT CGACACCACC ATAGACAGAA AGCGGTACAC CTCTACAAAG 4021 GAGGTCCTGG ACGCCACACT GATTCATCAG TCAATTACGG GGCTCTATGA AACAAGAATC 4081 GACCTCTCTC AGCTCGGTGG AGAC SEQ ID NO: 9: amino acid sequence of dead Cas9 DNA binding protein (1368 amino acids) 1 MDKKYSIGLA IGTNSVGWAV ITDEYKVPSK KFKVLGNTDR HSIKKNLIGA LLFDSGETAE 61 ATRLKRTARR RYTRRKNRIC YLQEIFSNEM AKVDDSFFHR LEESFLVEED KKHERHPIFG 121 NIVDEVAYHE KYPTIYHLRK KLVDSTDKAD LRLIYLALAH MIKFRGHFLI EGDLNPDNSD 181 VDKLFIQLVQ TYNQLFEENP INASGVDAKA ILSARLSKSR RLENLIAQLP GEKKNGLFGN 241 LIALSLGLTP NFKSNFDLAE DAKLQLSKDT YDDDLDNLLA QIGDQYADLF LAAKNLSDAI 301 LLSDILRVNT EITKAPLSAS MIKRYDEHHQ DLTLLKALVR QQLPEKYKEI FFDQSKNGYA 361 GYIDGGASQE EFYKFIKPIL EKMDGTEELL VKLNREDLLR KQRTFDNGSI PHQIHLGELH 421 AILRRQEDFY PFLKDNREKI EKILTFRIPY YVGPLARGNS RFAWMTRKSE ETITPWNFEE 481 VVDKGASAQS FIERMTNFDK NLPNEKVLPK HSLLYEYFTV YNELTKVKYV TEGMRKPAFL 541 SGEQKKAIVD LLFKTNRKVT VKQLKEDYFK KIECFDSVEI SGVEDRFNAS LGTYHDLLKI 601 IKDKDFLDNE ENEDILEDIV LTLTLFEDRE MIEERLKTYA HLFDDKVMKQ LKRRRYTGWG 661 RLSRKLINGI RDKQSGKTIL DFLKSDGFAN RNFMQLIHDD SLTFKEDIQK AQVSGQGDSL 721 HEHIANLAGS PAIKKGILQT VKVVDELVKV MGRHKPENIV IEMARENQTT QKGQKNSRER 781 MKRIEEGIKE LGSQILKEHP VENTQLQNEK LYLYYLQNGR DMYVDQELDI NRLSDYDVAA 841 IVPQSFLKDD SIDNKVLTRS DKARGKSDNV PSEEVVKKMK NYWRQLLNAK LITQRKFDNL 901 TKAERGGLSE LDKAGFIKRQ LVETRQITKH VAQILDSRMN TKYDENDKLI REVKVITLKS 961 KLVSDFRKDF QFYKVREINN YHHAHDAYLN AVVGTALIKK YPKLESEFVY GDYKVYDVRK 1021 MIAKSEQEIG KATAKYFFYS NIMNFFKTEI TLANGEIRKR PLIETNGETG EIVWDKGRDF 1081 ATVRKVLSMP QVNIVKKTEV QTGGFSKESI LPKRNSDKLI ARKKDWDPKK YGGFDSPTVA 1141 YSVLVVAKVE KGKSKKLKSV KELLGITIME RSSFEKNPID FLEAKGYKEV KKDLIIKLPK 1201 YSLFELENGR KRMLASAGEL QKGNELALPS KYVNFLYLAS HYEKLKGSPE DNEQKQLFVE 1261 QHKHYLDEII EQISEFSKRV ILADANLDKV LSAYNKHRDK PIREQAENII HLFTLTNLGA 1321 PAAFKYFDTT IDRKRYTSTK EVLDATLIHQ SITGLYETRI DLSQLGGD SEQ ID NO 12. CFTR Splice Acceptor 1 DNA nucleotide sequence 25 bp. This sequence corresponds to the nucleotide sequence between the left and right ITRs in the vector shown in FIG.4. ctgacctcttctcttcctcccacag SEQ ID NO 13. CFTR Splice Acceptor 2 DNA nucleotide sequence 89 bp. This sequence corresponds to the nucleotide sequence between the left and right ITRs in the vector shown in FIG.5. cccccccttttttttataaaattgtatgttatatacatatctcctgtatgtgacccatgtgcttatgac tctatttctcatgtgtttag SEQ ID NO 14. CFTR Splice Acceptor 3 DNA nucleotide sequence 436 bp. This sequence corresponds to the nucleotide sequence between the left and right ITRs in the vector shown in FIG.6. GCAAGAGTTCCAGCCGGGCTATTTACTTTTGTAAAACTTTATGGTTTGTGGAAAACAAATGTTTTTGAA CATTTAAAAAGTTCAGATGTTAAAAAGTTGAAAGGTTAATGTAAAACAATCAATATTAAAGAATTTTGA TGCCAAAACTATTAGATAAAAGGTTAATCTACATCCCTACTAGAATTCTCATACTTAACTGGTTGGTTA TGTGGAAGAAACATACTTTCACAATAAAGAGCTTTAGGATATGATGCCATTTTATATCACTAGTAGGCA 60 DB1 / 147031523.8 SAL-017PC / 126933-5017 GACCAGCAGACTTTTTTTTATTGTGATATGGGATAACCTAGGCATACTGCACTGTACACTCTGACATAT GAAGTGCTCTAGTCAAGTTTAACTGGTGTCCACAGAGGACATGGTTTAACTGGAATTCGTCAAGCCTCT GGTTCTAATTTCTCATTTGCAG SEQ ID NO: 11: amino acid encoding the human CFTR protein MQRSPLEKASVVSKLFFSWTRPILRKGYRQRLELSDIYQIPSVDSADNLSEKLEREWDRELAS KKNPKLINALRRCFFWRFMFYGIFLYLGEVTKAVQPLLLGRIIASYDPDNKEERSIAIYLGIGLCL LFIVRTLLLHPAIFGLHHIGMQMRIAMFSLIYKKTLKLSSRVLDKISIGQLVSLLSNNLNKFDEGLA LAHFVWIAPLQVALLMGLIWELLQASAFCGLGFLIVLALFQAGLGRMMMKYRDQRAGKISERL VITSEMIENIQSVKAYCWEEAMEKMIENLRQTELKLTRKAAYVRYFNSSAFFFSGFFVVFLSVL PYALIKGIILRKIFTTISFCIVLRMAVTRQFPWAVQTWYDSLGAINKIQDFLQKQEYKTLEYNLTT TEVVMENVTAFWEEGFGELFEKAKQNNNNRKTSNGDDSLFFSNFSLLGTPVLKDINFKIERGQ LLAVAGSTGAGKTSLLMMIMGELEPSEGKIKHSGRISFCSQFSWIMPGTIKENIIFGVSYDEYRY RSVIKACQLEEDISKFAEKDNIVLGEGGITLSGGQRARISLARAVYKDADLYLLDSPFGYLDVLT EKEIFESCVCKLMANKTRILVTSKMEHLKKADKILILHEGSSYFYGTFSELQNLQPDFSSKLMG CDSFDQFSAERRNSILTETLHRFSLEGDAPVSWTETKKQSFKQTGEFGEKRKNSILNPINSIRK FSIVQKTPLQMNGIEEDSDEPLERRLSLVPDSEQGEAILPRISVISTGPTLQARRRQSVLNLMT HSVNQGQNIHRKTTASTRKVSLAPQANLTELDIYSRRLSQETGLEISEEINEEDLKECFFDDME SIPAVTTWNTYLRYITVHKSLIFVLIWCLVIFLAEVAASLVVLWLLGNTPLQDKGNSTHSRNNSY AVIITSTSSYYVFYIYVGVADTLLAMGFFRGLPLVHTLITVSKILHHKMLHSVLQAPMSTLNTLKA GGILNRFSKDIAILDDLLPLTIFDFIQLLLIVIGAIAVVAVLQPYIFVATVPVIVAFIMLRAYFLQTSQ QLKQLESEGRSPIFTHLVTSLKGLWTLRAFGRQPYFETLFHKALNLHTANWFLYLSTLRWFQM RIEMIFVIFFIAVTFISILTTGEGEGRVGIILTLAMNIMSTLQWAVNSSIDVDSLMRSVSRVFKFID MPTEGKPTKSTKPYKNGQLSKVMIIENSHVKKDDIWPSGGQMTVKDLTAKYTEGGNAILENIS FSISPGQRVGLLGRTGSGKSTLLSAFLRLLNTEGEIQIDGVSWDSITLQQWRKAFGVIPQKVFIF SGTFRKNLDPYEQWSDQEIWKVADEVGLRSVIEQFPGKLDFVLVDGGCVLSHGHKQLMCLA RSVLSKAKILLLDEPSAHLDPVTYQIIRRTLKQAFADCTVILCEHRIEAMLECQQFLVIEENKVRQ YDSIQKLLNERSLFRQAISPSDRVKLFPHRNSSKCKSKPQIAALKEETEEEVQDTRL (SEQ ID NO: 11) SEQ ID NO: 304: E2C zinc finger (alone) – nucleotide sequence Ggaggtctcgaaccaggcgagaagccttatgcctgtcctgagtgtggcaaatccttctcaagaaaagactctctggttagacaccagagaacacatacaggg gagaaaccctataaatgccccgaatgcggaaagtccttttcccagagcggcgatctccggaggcatcagagaactcatacaggcgagaaaccatataagtg ccccgagtgtgggaaatccttttccgattgtagagacctggccagacatcaaaggacacatacaggcaagaagaccggaggt SEQ ID NO: 305: E2C zinc finger (alone) – amino acid sequence Gglepgekpyacpecgksfsrkdslvrhqrthtgekpykcpecgksfsqsgdlrrhqrthtgekpykcpecgksfsdcrdlarhqrthtgkktgg This disclosure is further illustrated by the following non-limiting examples. EXAMPLES Example 1: In Vitro Studies of CFTR Transgene Insertion into Endogenous CFTR Intron 1 Vectors will be bioengineered to target specific CFTR introns. Constructs that target site-specific TTAA sequences were used to target TTAA sites within CFTR intron 1. Engineered excision competent / target binding defective helper enzyme with site-specific DNA binders (e.g., 61 DB1 / 147031523.8 SAL-017PC / 126933-5017 without limitation, TALEs, Zinc fingers, dCasX) were fused with the helper enzyme. The engineered fusion protein inserted at the endogenous CFTR intron 1 contains CFTR exons 2 to 27 with the splice acceptor sites (SEQ. ID. NO.12, 13, or 14) to overcome all mutation in the endogenous exons 2-27. FIG.1 depicts a non-limiting schematic representation of the present approach to gene therapy. A lipid nanoparticle (LNP) delivers a donor DNA, i.e., CFTR gene, to the basal cell layer in the lung. The RNA helper enzyme integrates the CFTR2-27 transgene into an endogenous CFTR intron 1 site. FIG.2 depicts a schematic representation of the CFTR locus. The CFTR locus has important functional elements and complex regulation including airway-selective enhancer elements: -35 kb, -44 kb; intestinal-selective enhancer elements: 185+10kb (intron 1), 1811+0.8kb (intron 11); insulator elements: -20.9 kb, +15.6 kb; Topologically Associated Domains (TAD): -80.1 kb, +48.9 kb; weak CFTR promoter; and other key DNase I hypersensitive sites. Taken from Swahn H & Harris A, Genes 2019;10(3):235. FIG.3A, FIG.3B, and FIG.3C depicts schematic representations of RNA helper and DNA donor constructs. FIG. 3A shows an RNA helper construct using an extrinsic DNA binding domain (e.g., without limitation, dCas, chimeric dCasX molecules, TALE, Zinc finger) to target a specific TTAA site in CFTR intron 1. Dimerization enhancers (e.g., without limitation, SH3) and internal insertion of peptides to enhance non-covalent fusion of synthetic DNA binders (e.g., without limitation single-chain variable fragment (scFv) antibodies, monobodies, and nanobodies), may be used to improve targeting efficiency. FIG.3B shows DNA construct using a reporter gene (e.g., without limitation, GFP or luciferase) to test for site specific CFTR integration and lipid nanoparticle (LNP) targeting. FIG.3C shows a CFTR donor DNA containing helper-specific inverted terminal repeats (ITRs) flanking the open reading frame of CFTR exons 2-27 and a 5’-splice acceptor site (e.g. without limitation, SEQ ID NO: 12, 13, and 14) and a late SV40 poly(A) signal to terminate transcription. In vitro cell molecular studies to assess site-specific integration. The CFTR gene containing DNA donor and enzyme helper constructs generated above were used to transfect wild type (WT) and mutant (G542X) primary human bronchial epithelial cells (HBE) to identify the targeting constructs with the best integration efficiency, as measured, for instance, by GFP expression, qPCR, and western blot. In vitro readouts to determine restoration of CFTR function. Functional restoration of CFTR will be measured by molecular and phenotypic read-outs, including, without limitation, RTqPCR, western blotting, and functional assays of CFTR correction [e.g., transepithelial chloride conductance assay (TECC)] in isogenic human CF cell lines. Sequencing analysis to determine intronic targeting and off-target effects. Sequencing of the CFTR intron 1 transgene insertion site, and whole genome sequencing by methods. Methods such as, without limitation, LAM-PCR, Cas9-targeted sequencing adaptor ligation and long-read sequencing, and 62 DB1 / 147031523.8 SAL-017PC / 126933-5017 Tagmentation-Assisted PCR (tag-PCR) will be used to determine the intronic location of the CFTR transgene and off-target integrations. Example 2: Identification of LNPs that Target Pulmonary Basal Stem Cells via Inhalation and Intravenous by High- Throughput LNP Screening LNP formulation of CFTR and Reporter Constructs. Formulations capable of delivering DNA and mRNA to the tissues driven by two indications (hepatocytes for inherited lipid disorders and retina for inherited macular degeneration) have been developed previously through both rational design and empirical screening of a large set of parameters to identify formulations that are potent, well tolerated, and stable. Similar strategy will be used to identify LNP formulations with favorable biophysical characteristics, such as DNA and RNA encapsulation, size, and charge. Development of LNPs that target to lung. The LNP formulations developed above will be screened in vitro and in vivo using models for biodistribution and expression in CF-relevant cells in the lung via intravenous (IV) and inhalation routes. Reporter donor constructs will be employed for high throughput in vivo screening, which will allow for rapid iterative improvement in LNP characteristics. In addition to LNP development, alternate delivery technologies, such as a polymeric based nano- particle system, will be evaluated. Identification of LNPs that deliver to pulmonary basal cells. Targeting agents capable of promoting uptake into pulmonary basal stem cells via both IV and pulmonary routes will be identified through various experimentations. Through existing methods, these targeting agents will be decorated onto the surface of lead lung delivery LNPs. The targeted LNP formulations will be screened using a luciferase or GFP reporter plasmid. Determination and evaluation of lead formulations. Lead compositions selected in the above experiments will be engineered for CFTR plasmid and helper enzyme mRNA encapsulation efficiency, size, charge, etc. The engineered formulation(s) will be evaluated in air-liquid interface (ALI) cultures of primary HBE and isogenic, homozygous CF cell lines, for CFTR gene integration and expression. Develop a stable and scalable manufacturing process. Lead LNP formulations will be tested for penetration across cellular and mucus barriers. Cellular uptake will be tested in ALI primary HBE cultures to confirm the cellular uptake of LNPs decorated with mucus-penetrating peptides. The stability of lead LNP formulations will be tested in sputum specimens from CF patients, and expected to be able to withstand nebulization, multiple freeze-thaws, frozen, and refrigerated conditions. These formulations may also be engineered and scaled up to the support animal studies outlined below. 63 DB1 / 147031523.8 SAL-017PC / 126933-5017 Example 3: Phenotypic and Genotypic Correction of Cystic Fibrosis (CF) Animal Models Using Genome Engineering Molecular assessment after genome engineering. Initially, cftr− / − mouse model will be used to test lead LNP formulations. In vivo CFTR expression (e.g., without limitation, by RTqPCR and western blot), CFTR transgene copy number (ddPCR), CFTR intron 1-specific integration (Cas9-guided nanopore sequencing), and whole genome sequencing in basal cells, and other non- ciliated pulmonary airway cells, will be assessed for on-target and off-target integration. Phenotypic evaluation after genome engineering. Since murine models do not adequately mirror the pathophysiology of human CF, cftr- / - ferrets with tissue- corrected CFTR gene expression under the control of the iFABP gene promoter to prevent meconium ileus4 will be used as disease model. The cftr- / - ferret model will be treated with selected lead candidate to show CFTR transgene expression in basal cells and improvements in airway pathology. Example 4: Correction of Cystic Fibrosis (CF) in Patients Using Genome Engineering An objective of this study will be to compare the effectiveness of genome engineering method of the present disclosure in patients with cystic fibrosis (CF). The study will insert a large piece of healthy CFTR DNA at a precise location within the CFTR gene, which could enable the expression of a functional CFTR protein in essentially all individuals with CF. Such approach is expected to be effective regardless of the specific mutation(s) in the CFTR gene found in a given patient. Patients with various mutations in the CFTR genes will be compared before and after undergoing genome engineering method of the present disclosure. It is expected that the method will be able to restore functional CFTR proteins regardless of the specific mutations an individual patient carries. Example 5: Efficiency and specificity of integration Integration frequencies were measured using an element encoding for a fluorescent or antibiotic resistance protein (FIG.13). Next, element integration into the TTAA after transfection of the CFTR transgene-containing donor DNA and mRNA encoding the DBD and helper enzyme fusion were evaluated using a variety of strategies. For instance, one method to identify integrations into the chosen site was performed by PCR-amplification of genomic DNA between primers that bind to the integrating element and various positions on the target DNA followed by gel electrophoresis. The length of the amplicons approximately located the position of integration and the sequence of the amplicons which revealed their exact insertion proteins. FIG.7A and FIG.7B depict two AT-rich regions of intron 1 of the CFTR gene. The zinc finger and TALE sequences are in TABLE 1, TABLE 2, TABLE 3, and TABLE 4. The circle in FIG.7A shows the TTAA position targeted by the zinc finger 50. The circle in FIG.7B show the TTAA position targeted by the Z498 and Z504 as well as TALE pairs in the “IN” orientation ( ^ ⇐). 64 DB1 / 147031523.8 SAL-017PC / 126933-5017 FIG.8 depicts the results of GFP reporter assays using helper enzymes fused to various zinc fingers (ZF), donor, and plasmid reporters containing CFTR intron 1 regions A and B. HEK293 cells are seeded at 60-70% confluency 24hrs before the transfection. Helper 600ng, donor 300ng, reporter plasmid 300ng are transfected using the TransIT 2020 (MIRUSBIO, Madison, WI) according to manufacturer’s protocol. Briefly, 100ul OptiMEM and 1.2 ug DNA is added to a 1.5ml Eppendorf tube and vortexed and centrifuged briefly. TransIT 2020 (3.6ul) is added to all samples and incubated for 15 min. After 72hrs the half of the cell pellets are analyzed by Amplicon-seq the remaining cells using an Attune FACS machine with auto plate loader and gated for singlets and GFP. The percent of GFP positive cells are reported. The x-axis labels show the six finger ZF numbers in TABLE 1 that correspond to the CFTR regions A and B (hg38, chr7:117488023-117489022). The y-axis shows the percent of targeted inserts that show GFP expression at TTAA sites in the reporter plasmid. FIG.9 depicts the PCR results using a plasmid reporter assay in HEK293 cells using helper enzymes fused to various zinc fingers (ZF) and TALEs, donor, and plasmid reporters containing CFTR intron 1 regions A and B. The 1.5% agarose gel shows the expected PCR products using primers within the donor ITRs and outside the plasmid reporter integration site. FIG.9A shows the arrows pointed to the expected amplicons after donor insertion at the target TTAA for zinc finger-helper fusions to Z50 (Region A, 444 bp), Z498 (Region B, 625 bp) and Z504 (Region B, 625 bp) (sequences of Zinc fingers can be found in TABLE 1). FIG.9B shows the PCR results using TALE- helper fusions pairs to target the same TTAA. The 1.5% agarose gel shows the expected amplicon size (Region B, 475 bp) after donor insertion at the target TTAA for helper fusions to TALES. The spacing between the target TTAA and the TALE recognition sequences varied between 13 bp and 19 bp and only targeted when the TALE was in the “IN” orientation. For example, L-TALE2-IN was 15 bp 5’ to the target TTAA, R-TALE1-IN was 13 bp 3’ to the target TTAA, L-TALE3-IN was 19 bp 5’ to the target TTAA, R-TALE2-IN was 16 bp 3’ to the target TTAA, R- TALE3-IN was 18 bp 3’ to the target TTAA. FIG.10A, FIG.10B, FIG.10C, and FIG.10D depicts Amplicon-seq data using the cell pellets from the GFP reporter assay. The cell pellets were prepared for lysis using Viagen DirectCell (Los Angeles, CA) according to manufacturer’s protocol. Proteinase K powder (0.4mg / mL) and buffer (90 uL) was added to each pellet, incubated for 3hrs at 55°C rotating and heat inactivated for 45min at 85°C. One uL of the lysate was used as a genomic DNA template. A mastermix using KOD ONE BLUE (10uL) (Millipore Sigma, St, Louis, MO), water (7.8uL).10 uM each primer (0.6uL)(forward primer: 5’-GGGAGGATTGGGAAGACAAT-3’ (SEQ ID NO: 17); reverse primer: 5’- GCCCGCGCCATCTTATATTTTG-3’ (SEQ ID NO: 18)) was amplified using a ‘hotstart’ at 95°C for 30sec followed by 32 cycles of denaturation at 98°C for 10sec, annealing at 60°C for 5sec, and extension at 68°C for 10sec. Nine uL was electrophoresed on a 1.5% gel to verify amplification and the remaining 11 uL was cleaned using DNA clean and concentrator (Zymo Research, Irvine, CA) according to manufacturer’s protocol. The amplified DNA was quantified, and the samples were mixed with unique barcodes. DNA was diluted to 20ng / uL, and Illumina-based amplicon sequencing was performed at GeneWiz (South Plainfield, NJ). The data was uploaded to Watershed Bioinformatics (Cambridge, MA) custom amplicon-seq software. The software output determined the flanking 65 DB1 / 147031523.8 SAL-017PC / 126933-5017 sequence, position on reporter, number of reads, and percent insertion at each TTAA site. FIG.10A shows the TTAA position (bold) where most insertions occurred at specific CFTR intron 1 sites and the location of the nearby (12bp – 18 bp) ZF binding sites for ZF 654, 504 and 498. FIG.10B, FIG.10C, and FIG.10D show the number of reads at TTAA sites within the plasmid reporters containing CFTR intron 1 regions A and B. All three helper fusions to CFTR-specific ZF showed >90% targeting within 400 bp of the target TTAA. FIG.11 depicts a reporter assay in Saccharomyces cervisiae (yeast) to detect targeting by helper proteins fused to synthetic DNA binding proteins (e.g., ZF and TALEs). The URA3 gene flanked by donor ends was cloned into the chr VIII in yeast. A landing pad with a TTAA flanked by the synthetic DNA binding proteins was cloned into chr IX in yeast. A plasmid containing DNA helper with synthetic binding proteins are transformed into the yeast using Frozen-EZ Yeast Transformation II Kit (Zymo Research, Irvine, CA). Transposition is detected when the donor (URA3A first gene half) is transcribed and spliced to restore a functional URA3A gene. The yeast that are targeted with the donor insert at a specific TTAA will produce uracil and grow on uracil deficient media. Uracil is an essential nutrient for yeast and untargeted yeast will not grow on uracil deficient media. FIG.12 depicts a 1.5% agarose gel and PCR amplification strategy to identify yeast with targeted integration events at the intended TTAA. Primers were designed flanking the landing pad. Short PCR fragments did not include the donor insert while longer amplicons showed specific targeting to a TTAA that was 19 base pairs from the intended site. Sequencing was performed to confirm integration at the targeted site. Example 6: Restoration of CFTR function in primary HBE and isogenic CFF cell lines Transfections of primary HBE cells. After identifying a helper RNA (FIG.3A) for targeting CFTR intron 1, transfection of primary human bronchial epithelial (HBE) cells were initially tested, the donor DNA shown in FIG.3B are used to detect GFP expression. After establishing CFTR intron 1 targeting in HEK293 cells, helper RNA (FIG.3A) with the Donor DNA construct with CFTR2-27(FIG.3C) will be used to transfect HBE. Seventy-two hours after transfection the HBE cells were analyzed by flow cytometry and the percent of GFP expressing cells established the baseline transfection efficiency. HBE cells were cultured for 2 weeks and passaged 2 to 3 times per week. Flow cytometry were used to determine the percent of GFP expressing cells to measure the integration efficiency at 2 weeks. The final integration efficiency were determined by dividing the 2-week integration efficiency by the baseline. Genomic DNA from cell cultures with targeted integrants were analyzed by PCR using CFTR intron 1 primers flanking the targeted TTAA site and primers in exon 2, exon 27, right or left ITRs of the transgene. Targeted cells were sequenced using the methods known in the art. Transfection of isogenic, homozygous CF cell lines. FIG.17A shows a graph of RT-qPCR results using triplicate samples to determine the ΔCq value of WT versus G542X mutant cells when varying RNA template amounts were used in the assay. The ΔCq value was calculated as the difference between the average Cq value of CFTR and the average of the Cq values of a reference gene 66 DB1 / 147031523.8 SAL-017PC / 126933-5017 (TBP). The results showed the engineered range for the RNA template in the RTqPCR reaction is between 0.08- 0.16 ng when used in the One Step RT-qPCR System (BHioRad). FIG.17B shows a Western blot using a CFTR antibody with a ladder, loading control (NA+ / K+=ATPase), and protein from wild-type (WT) and G54216HBE cells. The lower band is a loading control (NA+ / K+=ATPase), the middle band, B, represents a partially glycosylated, non- functional CFTR, and the top band, C, a mature, fully functional CFTR. Example 7: Development of LNPs that target the lung Various experiments for lung delivery will be carried out to identify LNP formulations that accumulate in the lung following intravenous and inhalation routes of administration. Lead formulations will be used as a starting point to develop LNPs capable of delivering the CFTR payload to pulmonary basal cells through either route of administration. The efficacy of LNP formulations will be evaluated primarily in vivo. To start, a reporter plasmid of equal size to the CFTR plasmid will be delivered as a screening tool to evaluate potency and biodistribution of LNP formulations in mice. For lead formulations demonstrating acceptable potency and biodistribution profiles, the CFTR construct will be delivered and quantified for the degree of expression and gene integration. Example 8: Development of zinc fingers and TALEs targeting CFTR intron 1 FIG.9 depicts the PCR results using a plasmid reporter assay in HEK293 cells using helper enzymes fused to various zinc fingers (ZF) and TALEs, donor, and plasmid reporters containing CFTR intron 1 regions A and B. The 1.5% agarose gel shows the expected PCR products using primers within the donor ITRs and outside the plasmid reporter integration site. FIG.9A shows the arrows pointed to the expected amplicons after donor insertion at the target TTAA for zinc finger-helper fusions to Z50 (Region A, 444 bp), Z498 (Region B, 625 bp) and Z504 (Region B, 625 bp). FIG.15A and FIG.15B depict 2.0% agarose gels showing 6-finger zinc fingers and 3-finger zinc fingers targeting CFTR intron in a plasmid reporter assay. FIG.15A compares 6F ZF498, 6F ZF504, 6F ZF498 + ZF504, 3F ZF498, 3F ZF504, 3F ZF498 + ZF504 targeting of CFTR intro 1 (Table 1). FIG.15B compares 6F ZF50 and 3F ZF50 targeting of CFTR intro 1. Zinc fingers were designed to two regions of CFTR intron 1 in both 6-finger and 3-finger format in the plasmid reporter assay for evidence of targeting. Six finger ZnFs showed best targeted integration with reduced background. FIG.16 depicts 2.0% agarose gels showing TALES designed in the ‘IN’ orientation targeting CFTR intron 1 in the plasmid reporter assay. TALE binders flanking the TTAA site targeted by ZF498 and ZF504 were designed and tested. TALEs in the ‘IN’ orientation (left) outperformed TALEs in the ‘OUT’ (right) orientation (Table 1). TALES with “IN” orientation show targeted integration with reduced background. FIG.13 depicts the integration efficiency in mutant (G542X) HBE of a GFP donor element targeted to CFTR intron 1 using helper-fusions (TALE and Zinc fingers). HBE cells were transfected and evaluated at baseline and 2 weeks later. Flow cytometry was used to determine the percent of GFP expressing cells to measure the integration 67 DB1 / 147031523.8 SAL-017PC / 126933-5017 efficiency at 2 weeks. The final integration efficiency was determined by dividing the 2-week integration efficiency by the baseline. FIG.14 depicts a 2.0% agarose gel after transfection of the donor element in FIG.3C (CFTR exon 2-27) with different splice acceptor sites and a ZF504 helper fusion using a plasmid reporter assay in HEK293 cells. PCR primers were designed to identify the proper insertion of exons 2-27 in CFTR intron 1. The expected amplicon size for proper splicing was 630 bp. The gel shows that CFTR exon 2-27 was splice in the proper orientation in intron 1 of the endogenous CFTR gene by using a helper-Z504 fusion enzyme. Example 9: A Programmable Mammalian Transposon Achieves Accurate Integration of a CFTR2-27Superexon in Human Bronchial Epithelial Cells Introduction of large therapeutic genes to targeted sites under endogenous regulatory control is highly desired in diseases such as Cystic Fibrosis (CF). Recombination systems using DNA double strand break (DSB)-induced homology-directed repair have limitations (e.g., hazardous DSBs, gene cargo size, off-target insertions). Partial replacement of the CF transmembrane regulator gene (CFTR2-27) in intron 1 could provide a one-time, curative treatment that restores CFTR physiological expression levels and corrects >97% of CFTR downstream mutations. The example shows site-specific insertion of CFTR2-27cDNA in intron 1 using a bioengineered mammalian transposase derived from Myotis lucifugus (bMLT). This process achieved on-target integration, proper splicing, and adequate expression levels without DSBs, chromothripsis, indels, or rearrangements. The example achieved on-target integration into CFTR intron 1 with minimal off-target integrations. The programmable bioengineered mammalian transposase bMLT directed targeted insertion of a large cargo gene (12kb) from a non-viral plasmid donor into a chosen TTAA site in the human genome with minimal off-target insertions (FIG.18). Integration occurred in the absence of double stranded breaks in the genome, chromothripsis, indels, or rearrangements. The mammalian transposase was well-adapted to function at high mammalian cell growth temperatures. Targeted insertion, achieved by the addition of programmable DNA binders and enzyme mutations which enhanced fusion dependence and decreased off-target insertions, allowed cargo expression by native regulatory elements. The amino acid sequence of bMLT was modified to make it hyperactive and covalently fused a zinc finger (ZnFCFTR- bMLT) designed to recognize sequences near a CFTR intron 1 target site. The example used a “find and cut-and- transfer” (FiCAT) reporter assay to measure targeted integrations. DNA donor plasmids contained a splice acceptor followed by either a GFP reporter or tagged CFTR2-27, a polyadenylation sequence, flanking insulators, and bMLT terminal inverted repeats. The FiCAT assay was performed by lipofecting HEK293T cells with ZnFCFTR-bMLT, a GFP donor plasmid, and a CFTR intron 1 “landing pad reporter” plasmid (pCFTR) to analyze targeting efficiency of various bMLT mutants (n≥3). To assess CFTR intron 1-specific targeting efficiency in the human genome, published self-reporting transposon (SRT) methods were used to identify on- and off-target integrations in HEK293T cells lipofected with: a SRT donor plasmid and ZnFCFTR-bMLT (n≥8 per group). To quantitatively measure 68 DB1 / 147031523.8 SAL-017PC / 126933-5017 targeting efficiency, HEK293T cells were lipofected with tagged promoterless donor plasmid (GFP and CFTR-GFP donor conditions) and ZnFCFTR-bMLT (n=6). The promoterless donor plasmid was used to ensure GFP expression was controlled by endogenous CFTR regulatory elements. Targeting efficiency was estimated by quantitation of GFP positive cells via FACS followed by NGS sequencing confirmation. CFTR targeting was confirmed by PCR and Illumina NGS. Dual ZnF binding sites flanking the TTAA site synergistically increased targeted integration of desired cargo (FIG. 19A – 19C). FIG. 19A shows a schematic of FiCAT Reporter Assay. Gel electrophoresis of PCR products demonstrates targeted integration (FIG.19B). Next Generation Sequencing of integration showed a 2-fold increase in targeted integrations when the target TTAA site (arrow) was flanked by two ZnF binding sites (bottom graph) compared to a single ZnF binding site (top graph) (FIG.19C). Exc+ / IntreducedbMLT mutants increased targeted integration (FIG.20A – 20D). Mutation of non-specific target DNA binding domains of bMLT reduces integration (bars on the right) but not excision activity (bars on the left) (FIG. 20A). Next Generation Sequencing analysis showed increased targeted integration with various bMLT mutants at an engineered target site (bars on the right) within a reporter plasmid (FIG.20B). Flow analysis showed increased targeted integration at an engineered site (bars on the right) in the genome by various bMLT mutants using stable cell lines (FIG.20C). The bMLT mutants tested in FIG.20A-20C are shown below. Mut# Mutations FIG.20D shows ddPCR analysis of Targeted and Total Integration of 1st and 2nd Generation bMLT mutants. Further mutations to bMLT (brackets) reduce total integration 3-4-fold (bars on the right) without affecting targeted integration (bars on the left) compared to their parent mutants. Mut# Mutations Lanes 69 DB1 / 147031523.8 SAL-017PC / 126933-5017 ZnF-Mut1.1 E2C-S8P_C13R_D416N_Y281A_K334A_N335A_I332M 2 ZnF-Mut1.2 E2C-S8P C13R D416N Y281A K334A N335A I338M 3 ddton o xc+ / nte ucemut to nCFTRncreased spec c targetng at C ntron . ext Generation Sequencing demonstrated 40% higher targeted integration with ZnFCFTR-mut1 (bars labeled C) than ZnFCFTR-bMLT (bars labeled as B) and 63% higher targeted integration than unfused bMLT (bars labeled as A) (FIG.21A). SRT sequencing results showed a ~200-fold increase in targeted integration (bars on the left) and ~30-fold decrease in integrations elsewhere (bars on the right) (FIG.21B). Addition of ZnF binding sites flanking the cargo element in the donor plasmid increased overall integration. FIG. 22A showed a schematic of donor plasmids contain no flanking ZnF binding sites (top) or a ZnFCFTRbinding site flanking each ITR outside of the cargo (bottom). Flow analysis showed a ~2.5-fold increase in targeted integration by ZnFCFTR-bMLT when two flanking ZnFCFTR binding sites (bars on the right) are added to the cargo element (FIG. 22B). ZnFCFTR-mut1 bMLT showed targeted genomic integration of reporter and CFTR2-27 donors in HEK293T and human bronchial epithelial (HBE) cells modeling Cystic Fibrosis. Promoterless GFP and CFTR2-27-GFP plasmids with different splice acceptors were transfected alongside ZNFCFTR–mut1 bMLT into HEK293T cells under varying conditions (FIG.23A and FIG.23C). FIG.23B and FIG.23D showed FACS analysis of targeted integration in HEK293T cells. Cells transfected under engineered conditions displayed higher targeted integration (up to 10%) when compared to control. FIG.23E showed targeted integration in CFTR G542X 16HBEge cells. Cells were transfected with the donor shown in FIG.23A and various Exc+ / Intreducedmutants. FACS analysis showed an up to 4-fold increase in targeted integration compared to donor alone. The example achieved on-target integration of a CFTR2-27superexon into CFTR intron 1 in HEK293T cells and in a mutant bronchial epithelial cell line that mimics CF (CFTR G542X). Targeted integration was achieved with minimal off-target integrations. 70 DB1 / 147031523.8 SAL-017PC / 126933-5017 EQUIVALENTS While the disclosure has been described in connection with specific embodiments thereof, it will be understood that it is capable further modifications of and this application is intended to cover any variations, uses, or adaptations of the disclosure following, in general, the principles of the disclosure and including such departures from the present disclosure as come within known or customary practice within the art to which the disclosure pertains and as may be applied to the essential features herein set forth and as follows in the scope of the appended claims. Those skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation, numerous equivalents to the specific embodiments described specifically herein. Such equivalents are intended to be encompassed in the scope of the following claims. INCORPORATION BY REFERENCE All patents and publications referenced herein are hereby incorporated by reference in their entireties. The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. As used herein, all headings are simply for organization and are not intended to limit the disclosure in any manner. The content of any individual section may be equally applicable to all sections. 71 DB1 / 147031523.8

Claims

SAL-017PC / 126933-5017 CLAIMS What is claimed is:

1. A composition comprising a gene transfer construct, comprising: (a) a nucleic acid encoding a cystic fibrosis transmembrane conductance regulator (CFTR) transgene or a functional fragment thereof; and (b) a vector comprising one or more inverted terminal repeats (ITRs) or end sequences.

2. The composition of claim 1, wherein the gene transfer construct comprises DNA.

3. The composition of claim 1 or 2, wherein the gene transfer construct is codon optimized.

4. The composition of any one of claims 1 to 3, wherein the CFTR transgene is or comprises human CFTR exons 2 to 27 (CFTR2-27), or a functional fragment thereof.

5. The composition of claim 4, wherein the human CFTR exons 2 to 27, or a functional fragment thereof, is suitable for insertion into one or more loci of TABLEs A-B, optionally an endogenous CFTR intron 1 locus.

6. The composition of any one of claims 1-5, wherein the composition comprises a 5’-splice acceptor site.

7. The composition of any one of claims 1-6, wherein the composition comprises a late SV40 poly(A) signal.

8. The composition of claim 7, wherein the nucleic acid encoding CFTR transgene, or the functional fragment thereof comprises: a nucleotide sequence of SEQ ID NO: 10, or a variant having at least about 90%, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto; a nucleotide sequence of SEQ ID NO: 12, or a variant having at least about 90%, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto; a nucleotide sequence of SEQ ID NO: 13, or a variant having at least about 90%, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto; or a nucleotide sequence of SEQ ID NO: 14, or a variant having at least about 90%, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto.

9. The composition of claim 7, wherein the nucleic acid encoding CFTR transgene, or a functional fragment thereof comprises: an amino acid sequence of SEQ ID NO: 11 or a variant having at least about 90%, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto; or 72 DB1 / 147031523.8SAL-017PC / 126933-5017 an amino acid sequence of SEQ ID NO: 306, or a variant having at least about 90%, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto.

10. The composition of any one of claims 1-9 wherein the vector encodes from 5’ to 3’, a first ITR sequence, a CFTR transgene, and a second ITR sequence.

11. The composition of any one of claim 1-10, wherein the CFTR transgene further comprises a promoter, optionally wherein the CFTR transgene targets a safe harbor site.

12. The composition of any one of claims 1-10, wherein the CFTR transgene does not comprise a promoter, optionally wherein the CFTR transgene targets the CFTR gene.

13. The composition of any one of claims 1-12, wherein the vector is a DNA plasmid and optionally wherein the DNA plasmid comprises one or more insulator sequences that prevent or mitigate activation or inactivation of nearby genes.

14. The composition of any one of claims 1-13, wherein: (a) the ITRs or the end sequences are those of a mobile element, optionally comprising a TTAA repetitive sequence, and / or (b) the ITRs or the end sequences flank the nucleic acid encoding the CFTR transgene, or a functional fragment thereof.

15. The composition of claim 14, wherein the ITRs or the end sequences are selected from SEQ ID NO: 6 and SEQ ID NO: 7, or a variant thereof, optionally wherein the end sequences are selected from a sequence of SEQ ID NO: 6 or a variant thereof and a truncation of nucleotide 78-167, or positions corresponding thereto.

16. The composition of claim 14, wherein the TTAA repetitive sequence comprises ttTTAAaa (SEQ ID NO: 302) or taTTAAta (SEQ ID NO: 303) repetitive sequence.

17. The composition any one of claims 1-16, further comprising a nucleic acid construct encoding an enzyme capable of performing targeted genomic integration, optionally an mRNA encoding the enzyme.

18. The composition of any one of claims 1-17, wherein the enzyme is derived from Myotis lucifugus, Bombyx mori, Xenopus tropicalis, Trichoplusia ni, Rhinolophus ferrumequinum, Rousettus aegyptiacus, Phyllostomus discolor, Myotis myotis, Pteropus vampyrus, Pipistrellus kuhlii, Pan troglodytes, Molossus molossus, Homo sapiens, Eptesicus fuscus, Scalopus aquaticus, Pipistrellus pipistrellus, Crasionycteris thonglongyai, Microcebus murinus, Hipposideros galeritus, Hipposideros armiger, Murina feae, Megaderma lyra, Echinops telfairi, Balaenoptera bonaerensis, Delphinus leucas, Monodon monoceros, Kogia breviceps, Echinops telfairi, Daubentonia madagascarensis, Mizra coquereli, or one or more of the Tn1, Tn2, Tn3, Tn5, Tn7, Tn9, Tn10, Tn552, Tn903, Tn1000 / Gamma-delta, Tn / O, tnsA, tnsB, tnsC, tniQ, IS10, ISS, IS911, Minos, Sleeping beauty, piggyBac, Tol2, Mos1, Himar1, Hermes, Tol2, Minos, Tel, P- element, MuA, Ty1, Chapaev, transib, Tc1 / mariner, or Tc3 transposon system, or biologically active 73 DB1 / 147031523.8SAL-017PC / 126933-5017 fragments variants thereof, inclusive of hyperactive variants, and / or is an engineered version thereof and / or wherein the enzyme specifically recognizes the ITRs or the end sequences.

19. The composition of claim 18, wherein the enzyme is derived from Myotis lucifugus or Eptesicus fuscus.

20. A composition of any one of claims 1-19, wherein the enzyme comprises an amino acid sequence having at least about 80% sequence identity to SEQ ID NO: 1 and has an alanine residue at position 2 of SEQ ID NO: 1 or a position corresponding thereto.

21. The composition of claim 20, wherein the enzyme comprises an amino acid sequence of at least about 90% identity to SEQ ID NO:

1.

22. The composition of claim 20, wherein the enzyme comprises an amino acid sequence of at least about 93% identity to SEQ ID NO:

1.

23. The composition of claim 20, wherein the enzyme comprises an amino acid sequence of at least about 95% identity to SEQ ID NO:

1.

24. The composition of claim 20, wherein the enzyme comprises an amino acid sequence of at least about 98% identity to SEQ ID NO:

1.

25. The composition of claim 20, wherein the enzyme comprises an amino acid sequence of at least about 99% identity to SEQ ID NO:

1.

26. The composition of any one of claims 20-25, wherein the enzyme has one or more mutations which confer hyperactivity.

27. The composition of any one of claims 20-26, wherein the enzyme has one or more amino acid substitutions selected from S8X1, C13X2, and / or D416X3or substitutions corresponding thereto relative to SEQ ID NO:

1.

28. The composition of claim 27, wherein X1is selected from G, A, V, L, I and P, 29. The composition of claim 27, wherein X2is selected from K, R, and H.

30. The composition of claim 27, wherein X3is selected from N, Q, S, T, P, and C.

31. The composition of any one of claims 27-30, wherein: X1is P, X2is R and X3is N.

32. The composition of any one of claims 20-31, wherein the enzyme has at least one, or two, or three, or four, or five, or six, or seven, or eight substitutions at a position corresponding to Y281A, G285A, I332M, R333A, R333K, R333S, K334A, K334R, K334S, N335A, R336A, R336K, R336S, G337A, and I338M or substitutions corresponding thereto relative to SEQ ID NO:

1.

33. The composition of any one of claims 20-32, wherein at least one, or two, or three, or four, or five, or six, or seven, or eight substitutions at a position corresponding to S8P, C13R, D416N Y281A, G285A, I332M, 74 DB1 / 147031523.8SAL-017PC / 126933-5017 R333A, R333K, R333S, K334A, K334R, K334S, N335A, R336A, R336K, R336S, G337A, and I338M or substitutions corresponding thereto relative to SEQ ID NO:

1.

34. The composition of any one of claims 20-33, the helper enzyme comprises a substitution selected from TABLE C and / or TABLE D or a substitution at positions corresponding thereto relative to SEQ ID NO:

1.

35. The composition of any one of claims 20-33, wherein the helper enzyme comprises S8P_C13R_D416N_Y281A_K334A_N335A_I332M, S8P_C13R, S8P_C13R_D416N_Y281A_K334A_N335A, S8P_C13R_D416N_R333A, S8P_C13R_D416N_R333A_K334A, S8P_C13R_D416N_R333A_N335A, S8P_C13R_D416N_K334A_R336A, S8P_C13R_D416N_Y281A_K334A_N335A_I338M, S8P_C13R_D416N_Y281A_K334A_N335A_I332M_I338M, S8P_C13R_D416N_R333A_N335A_K334R, S8P_C13R_D416N_R333A_N335A_K334S, S8P_C13R_D416N_R333A_N335A_K334A, S8P_C13R_D416N_R333A_N335A_I338M, S8P_C13R_D416N_R333A_N335A_I332M_I338M, or substitutions corresponding thereto relative to SEQ ID NO:

1.

36. The composition of any one of claims 20-33, wherein the helper enzyme comprises E2C- S8P_C13R_D416N_Y281A_K334A_N335A_I332M, E2C-S8P_C13R, E2C- S8P_C13R_D416N_Y281A_K334A_N335A, E2C-S8P_C13R_D416N_R333A, E2C- S8P_C13R_D416N_R333A_K334A, E2C-S8P_C13R_D416N_R333A_N335A, E2C- S8P_C13R_D416N_K334A_R336A, E2C-S8P_C13R_D416N_Y281A_K334A_N335A, E2C- S8P_C13R_D416N_Y281A_K334A_N335A_I338M, E2C- S8P_C13R_D416N_Y281A_K334A_N335A_I332M_I338M, E2C-S8P_C13R_D416N_R333A_N335A, E2C-S8P_C13R_D416N_R333A_N335A_K334R, E2C-S8P_C13R_D416N_R333A_N335A_K334S, E2C-S8P_C13R_D416N_R333A_N335A_K334A, or substitutions corresponding thereto relative to SEQ ID NO:

1.

37. The composition of any one of claims 20-36, wherein the enzyme has an N-terminal deletion of about 2 to about 35 amino acids, or from about 2 to about 45 amino acids, or from about 2 to about 70 amino acids.

38. The composition of any one of claims 1-19, wherein the enzyme comprises an amino acid sequence of SEQ ID NO: 4 or 15.

39. The composition of any one of claims 1-19, wherein the nucleic acid that encodes the enzyme has a nucleotide sequence of SEQ ID NO: 5 or 16 or a codon-optimized form thereof.

40. The composition of any one of claims 1-39, wherein the enzyme has increased activity relative to an enzyme comprising an amino acid sequence of SEQ ID NO: 1, 4, 15, or functional equivalent thereof. 75 DB1 / 147031523.8SAL-017PC / 126933-5017 41. The composition of any one of claims 1-39, wherein the enzyme has excision activity.

42. The composition of any one of claims 1-39, wherein the enzyme has decreased integration activity.

43. The composition of any one of claims 1-39, wherein the enzyme has decreased integration activity relative to an enzyme comprising an amino acid sequence of SEQ ID NO: 1, 4, 15, or functional equivalent thereof.

44. The composition of any one of claims 1-39, wherein the enzyme has increased excision activity relative to an enzyme comprising an amino acid sequence of SEQ ID NO: 1, 4, 15 or functional equivalent thereof.

45. The composition of any one of claims 1-39, wherein the enzyme comprises a targeting element.

46. The composition of claim 45, wherein the targeting element binds a target site selected from any one of the targets sites of Tables 1-4.

47. The composition of claim 45, wherein the targeting element binds target sites in CFTR intron 1.

48. The composition of claim 47, wherein the target sites in CFTR intron 1 comprise a nucleotide sequence having at least about 80%, or at least about 90%, or at least about 93%, or at least about 95%, or at least about 98%, or at least about 99% sequence identity to SEQ ID NO:

313.

49. The composition of claim 45, wherein the targeting element binds a target site in a genomic safe harbor site (GSHS).

50. The composition of claim 45, wherein the targeting element binds a genomic sequence comprising 20 nucleotides of the 5’ or 3’ end of a TTAA site in intron 1.

51. The composition of claim 50, wherein the TTAA site comprises ttTTAAaa (SEQ ID NO: 302) or taTTAAta (SEQ ID NO: 303) sequences.

52. The composition of claim 50 or 51, wherein the targeting elements bind upstream and / or downstream of the TTAA sites.

53. The composition of claim 52, wherein the targeting elements bind within about 10 base pairs, or about 12 base pairs, or about 14 base pairs, or about 16 base pairs, or about 18 base pairs, or about 20 base pairs, or about 22 base pairs or about 24 base pairs, or about 26, base pairs or about 28 base pairs, or about 30 base pairs of the TTAA sites.

54. The composition of any one of claims 1-39, wherein the control is a composition comprising an enzyme comprising an amino acid sequence of SEQ ID NO: 1 or a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 2 or a codon-optimized form thereof.

55. The composition of any one of claims 1-35 or 37-54, wherein the targeting element is or comprises one or more of a Cas enzyme, which is optionally catalytically inactive and which is optionally associated with a Zinc finger, guide RNA (gRNA), transcription activator-like effector (TALE) DNA binding domain (DBD), catalytically inactive transcription factor, catalytically inactive nickase, a transcriptional activator, a 76 DB1 / 147031523.8SAL-017PC / 126933-5017 transcriptional repressor, a recombinase, a DNA methyltransferase, a histone methyltransferase, and a paternally expressed gene 10 (PEG10).

56. The composition of claim 55, wherein the targeting element is inserted after residue S60, or after residue E70, or after residue L80, or after residue N90, or after residue D100, relative to SEQ ID NO: 1, optionally by way of a linker.

57. The composition of claim 56, wherein the targeting element is inserted immediately after residue G66 relative to SEQ ID NO: 1, optionally by way of a linker.

58. The composition of any one of claims 55-57, wherein the targeting element comprises a Zinc finger, optionally a 6 finger Zinc finger or a 3 finger Zinc finger.

59. The composition of claim 58, wherein the Zinc finger is selected from TABLE 1, TABLE 2, or TABLE 3.

60. The composition of claim 59, wherein the Zinc finger is a E2C Zinc finger.

61. The composition of claim 60, wherein the E2C Zinc finger comprises at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% identity to an amino acid sequence of SEQ ID NO: 305 or a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 304 or a codon-optimized form thereof.

62. The composition of any one of claims 58-61, wherein the Zinc finger binds a target site in CFTR intron 1.

63. The composition any one of claims 58-61, wherein the Zinc finger binds a target site in a genomic safe harbor site (GSHS).

64. The composition of any one of claims 55-57, wherein the targeting element comprises a TALE DBD.

65. The composition of claim 64, wherein the TALE DBD comprises one or more repeat sequences.

66. The composition of claim 65, wherein the TALE DBD comprises about 14, or about 15, or about, 16, or about 17, or about 18, or about 18.5 repeat sequences.

67. The composition of claim 65 or claim 66, wherein the repeat sequences each independently comprises about 33 or 34 amino acids.

68. The composition of claim 67, wherein the repeat sequences each independently comprises a repeat variable di-residue (RVD) at residue 12 or 13 of the 33 or 34 amino acids, respectively.

69. The composition of claim 68, wherein the RVD recognizes one base pair in a target nucleic acid sequence.

70. The composition of claim 68 or claim 69, wherein the RVD recognizes a C residue in the target nucleic acid sequence and is selected from HD, N(gap), HA, ND, and HI.

71. The composition of claim 68 or claim 69, wherein the RVD recognizes a G residue in the target nucleic acid sequence and is selected from NN, NH, NK, HN, and NA. 77 DB1 / 147031523.8SAL-017PC / 126933-5017 72. The composition of claim 68 or claim 69, wherein the RVD recognizes an A residue in the target nucleic acid sequence and is selected from NI and NS.

73. The composition of claim 68 or claim 69, wherein the RVD recognizes a T residue in the target nucleic acid sequence and is selected from NG, HG, H(gap), and IG.

74. The composition of any one of claims 64-73, wherein the TALE DBD is selected from TABLE 4.

75. The composition of any one of claims 64-74, wherein the TALE DBD binds a target site in CFTR intron 1.

76. The composition of claim 64-74, wherein the TALE DBD binds a target site in a genomic safe harbor site (GSHS).

77. The composition of any one of claims 55-57, wherein the targeting element comprises a Cas9 enzyme associated with a gRNA or a CasX enzyme associated with a gRNA.

78. The composition of claim 77, wherein the Cas9 enzyme associated with a gRNA comprises a catalytically inactive dCas9 associated with a gRNA or an inactive CasX enzyme associated with a gRNA.

79. The composition of claim 78, wherein the catalytically inactive dCas9 comprises at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% identity to an amino acid sequence of SEQ ID NO: 9 or a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 8 or a codon-optimized form thereof.

80. The composition of any one of claims 55-57, wherein the targeting element comprises a Cas12 enzyme associated with a gRNA.

81. The composition of claim 80, wherein the targeting element comprises a catalytically inactive Cas12 associated with a gRNA, optionally wherein the catalytically inactive Cas12 is dCas12j or dCas12a.

82. The composition of claim any one of claims 55-57, wherein the targeting element comprises a Cas6, Cas7, or Cas8 enzyme associated with a gRNA.

83. The composition of any one of claims 55-82, wherein the targeting element comprises a nucleic acid binding component of a gene-editing system.

84. The composition of any one of claims 1-83, wherein the enzyme or variant thereof and the targeting element are connected.

85. The composition of claim 84, wherein the enzyme and the targeting element are fused to one another or linked via a linker to one another.

86. The composition of claim 85, wherein the linker is a covalent linker or a non-covalent linker.

87. The composition of claim 85, wherein the linker comprises less than about 25 amino acids or 75 nucleotides. 78 DB1 / 147031523.8SAL-017PC / 126933-5017 88. The composition of claim 85, wherein the linker comprises about 10 amino acids to about 20 amino acids or about 12 amino acids to about 15 amino acids, or about 30 nucleotides to about 60 nucleotides or about 36 nucleotides to about 45 nucleotides.

89. The composition of any one of claims 85-88, wherein the linker is substantially comprised of glycine (G) and serine (S) residues.

90. The composition of any one of claims 85-89, wherein the linker is or comprises (GSS)4or the linker is GS flanked on either side of a DNA binding domain, optionally TALE and ZnF.

91. The composition of claim 86, wherein the linker comprises an amino acid sequence of AKLAGGAPAVGGGPKAADKFAATGGS (SEQ ID NO: 8) or a variant thereof having a substitution or deletion.

92. The composition of any one of claims 85-91, wherein the linker connects the targeting element to the N- terminus of the helper enzyme or connects the targeting element within the helper enzyme.

93. The composition of claim 86, wherein the linker is a non-covalent linker.

94. The composition of claim 93, wherein the non-covalent linker comprises an epitope tag, optionally wherein the epitope tag comprises an ALFA tag.

95. The composition of claim 94, wherein the ALFA tag further comprises recombinant variable domains of heavy-chain-only antibodies (VHH) (NANOBODY).

96. The composition of any one of claims 86-95, wherein the linker is inserted after residue S60, or after residue E70, or after residue L80, or after residue N90, or after residue D100 relative to SEQ ID NO:

1.

97. The composition of claim 96, wherein the linker is inserted immediately after residue G66 relative to SEQ ID NO:

1.

98. The composition of any one of claims 1-97 wherein the enzyme or variant thereof is able to directly or indirectly cause transposition of a target gene.

99. The composition of any one of claims 1-98, wherein the enzyme or variant thereof is able to directly or indirectly interact and / or form a complex with one or more proteins or nucleic acids.

100. The composition of any one of claims 1-97, wherein a nucleic acid encoding the enzyme capable of targeted genomic integration by transposition comprises an intein, optionally NpuN (Intein-N) (SEQ ID NO: 423) and / or NpuC (Intein-C) (SEQ ID NO: 424), or a variant thereof.

101. The composition of claim 100, wherein the nucleic acid encodes the enzyme in the form of first and second portions with the intein encoded between the first and second portions, such that the first and second portions are fused into a functional enzyme upon post-translational excision of the intein from the enzyme. 79 DB1 / 147031523.8SAL-017PC / 126933-5017 102. The composition of claim 100 or claim 101, wherein the intein is suitable for linking the enzyme and the targeting element.

103. The composition of any one of claims 1-97, wherein a nucleic acid encoding the enzyme capable of targeted genomic integration by transposition comprises a dimerization enhancer.

104. The composition of claim 103, wherein the nucleic acid encodes the enzyme in the form of first and second portions with the dimerization enhancer encoded between the first and second portions, such that the first and second portions are fused into a functional enzyme upon post-translational excision of the dimerization enhancer from the enzyme.

105. The composition of claim 103 or claim 104, wherein the dimerization enhancer is suitable for linking the enzyme and the targeting element.

106. The composition of any one of claims 103-105, wherein the dimerization enhancer is selected from: a protein comprising a SH3 domain, biotin, avidin, or a rapamycin binder, optionally, wherein the rapamycin binder is FKBP12 or mTOR, or a variant thereof.

107. The composition of any one of claims 1-106, wherein the transgene comprises a nucleic acid sequence and a first and a second end sequences.

108. The composition of claim 107, wherein the transgene is flanked by the first and the second end sequences.

109. The composition of claim 107 or claim 108, wherein the end sequences are selected from nucleotide sequences of SEQ ID NO: 6 and / or SEQ ID NO: 7, or a nucleotide sequence having at least about 90% identity thereto.

110. The composition of any one of claims 107-109, wherein the end sequences include at least one repeat from a nucleotide sequence having at least about 90% identity to the nucleotide sequence of SEQ ID NO:

6.

111. The composition of claim 110, wherein the at least one repeat from the nucleotide sequence having at least about 90% identity to the nucleotide sequence of SEQ ID NO: 6 is positioned at the 5’ end of the transgene.

112. The composition of any one of claims 107-109, wherein the end sequences further comprise at least one repeat from a nucleotide sequence having at least about 90% identity to the nucleotide sequence of SEQ ID NO:

7.

113. The composition of any one of claims 107-109, wherein the at least one repeat from the nucleotide sequence having at least about 90% identity to the nucleotide sequence of SEQ ID NO: 7 is positioned at the 3’ end of the transgene. 80 DB1 / 147031523.8SAL-017PC / 126933-5017 114. The composition of any one of claims 1-113, wherein the enzyme or variant thereof is incorporated into a vector or a vector-like particle.

115. The composition of claim 114, wherein the vector or a vector-like particle comprises one or more expression cassettes.

116. The composition of claim 115, wherein the vector or a vector-like particle comprises one expression cassette.

117. The composition of claim 116, wherein the expression cassette further comprises the enzyme or variant thereof, the transgene, the end sequences, or a combination thereof.

118. The composition of claim 114, wherein the enzyme or variant thereof, the transgene, the end sequences, or a combination thereof are incorporated into one or more vectors or vector-like particles.

119. The composition of claim 114, wherein the enzyme or variant thereof, the transgene, the end sequences, or combination thereof are incorporated into a same vector or vector-like particle.

120. The composition of claim 114, wherein the enzyme or variant thereof, the transgene, the end sequences, or combination thereof is incorporated into different vectors or vector-like particles.

121. The composition of any one of claims 118-120, wherein the vector or vector-like particle is nonviral.

122. The composition of any one of claims 1-121, wherein the transgene does not comprise a promoter, optionally wherein the transgene targets the CFTR gene.

123. The composition of any one of claims 1-121, wherein the transgene is under the control of at least one tissue-specific promoter, optionally wherein the transgene targets a safe harbor site.

124. The composition of claim 123, wherein the at least one tissue-specific promoter is a single promoter.

125. The composition of claim 123, wherein the at least one tissue-specific promoter is under the control of a dual promoter or a tandem promoter.

126. The composition of any one of claims 1-125, wherein the transgene comprises peptides for linking fragments thereof.

127. The composition of claim 126, wherein the transgene is linked to polynucleotide comprising a sequence comprising a 5’-miRNA, a sense and antisense miRNA pair, and / or a 3’-miRNA.

128. The composition of any one of claims 1-127, wherein the composition comprises DNA, RNA, or both.

129. The composition of any one of claims 1-127, wherein the enzyme or variant thereof is in the form of RNA.

130. A host cell comprising the composition any one of claims 1-129.

131. The composition of any one of claims 1-130, wherein the composition is encapsulated in a lipid nanoparticle (LNP). 81 DB1 / 147031523.8SAL-017PC / 126933-5017 132. The composition of any one of claims 1-131, wherein the polynucleotide encoding the enzyme or variant thereof and the polynucleotide encoding the transgene are in the form of the same LNP, optionally in a co-formulation.

133. The composition of claim 131 or claim 132, wherein the LNP comprises one or more lipids selected from 1,2-dioleoyl-3-trimethylammonium propane (DOTAP), a cationic cholesterol derivative mixed with dimethylaminoethane-carbamoyl (DC-Chol), phosphatidylcholine (PC), triolein (glyceryl trioleate), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethylene glycol)-2000] (DSPE-PEG), 1,2-dimyristoyl-rac-glycero-3-methoxypolyethyleneglycol – 2000 (DMG-PEG 2K), and 1,2 distearol -sn- glycerol-3phosphocholine (DSPC) and / or comprising of one or more molecules selected from polyethylenimine (PEI) and poly(lactic-co-glycolic acid) (PLGA), N-Acetylgalactosamine (GalNAc), and SSOP LNPs.

134. A method for treating and / or mitigating cystic fibrosis (CF), comprising administering to a patient in need thereof a composition of any one of claims 1-133.

135. A composition comprising a gene transfer construct, comprising: (a) a nucleic acid encoding a cystic fibrosis transmembrane conductance regulator (CFTR) transgene or a functional fragment thereof, wherein the nucleic acid construct further comprises a nucleic acid construct encoding an enzyme capable of performing targeted genomic integration, optionally wherein the enzyme comprises an amino acid sequence having at least about 80%, or at least about 90%, or at least about 93%, or at least about 95%, or at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 1, and (b) a vector comprising one or more inverted terminal repeats (ITRs) or end sequences.

136. The composition of claim 135, wherein the enzyme comprises S8P_C13R_D416N_Y281A_K334A_N335A_I332M, or substitutions corresponding thereto relative to SEQ ID NO:

1.

137. The composition of claims 135 or 136, wherein the enzyme comprises a targeting element.

138. The composition of any one of claims 135-137, wherein the targeting element comprises a Zinc finger, optionally a 6 finger Zinc finger or a 3 finger Zinc finger.

139. The composition of claim 138, wherein the Zinc finger is a E2C Zinc finger.

140. The composition of claim 139, wherein the E2C Zinc finger comprises at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% identity to an amino acid sequence of SEQ ID NO: 305 or a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 304 or a codon-optimized form thereof. 82 DB1 / 147031523.8

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